Bolaamphiphilic compounds, compositions and uses thereof

ABSTRACT

Bolaamphiphilic compounds are provided according to formula I: 
       HG 2 -L 1 -HG 1   I
 
     where HG 1 , HG 2  and L 1  are as defined herein. Provided bolaamphiphilic compounds and the pharmaceutical compositions thereof are useful for delivering HIV active drugs into animal or human brain.

CROSS REFERENCES TO RELATED APPLICATIONS

This is a continuation-in-part of International ApplicationPCT/US13/57957, filed on Sep. 4, 2013, which claims priority to U.S.Patent Application 61/696,786, filed Sep. 2, 2012. This application alsoclaims the benefit of U.S. Patent Application 61/844,782, filed on Jul.10, 2013. The contents of each of the above-referenced applications areincorporated by reference herein.

FIELD

Provided herein are nanovesicles comprising bolaamphiphilic compounds,and complexes thereof with HIV drug molecules, and pharmaceuticalcompositions thereof. Also provided are methods of delivering HIV drugmolecules into the human brain and animal brain using the compounds,complexes and pharmaceutical compositions provided herein.

BACKGROUND

Many AIDS patients, after receiving Highly Active Anti RetroviralTreatment (HAART), do not have detectable HIV in the blood, but theycontinue to have measurable amounts of HIV in the central nervous system(CNS). It is probable that this persistent HIV in the CNS (called,neuro-HIV) [Spudich and Antses, 2011] is a major obstacle in eradicatingHIV in patients despite long-term HAART treatment [Gisslen et al, 2001;Highleyman, 2009, Varatharajan et al, 2009]. The incidence ofHIV-associated dementia (HAD) has been reduced significantly withlong-term HAART, but cognitive impairment is still measurable in as manyas 50% of treated patients [Highleyman, 2009]. The reason for thecognitive impairment is, presumably, the persistence of HIV in thecerebrospinal fluid (CSF) of treated patients, despite suppression ofthe serum viral loads by HAART. Thus, it appears that the brain servesas a reservoir for persistent HIV, and that this reservoir preventslong-term HAART from curing HIV patients and alleviating cognitivemalfunctions.

The brain is a highly specialized organ, and its sensitive componentsand functioning are protected by a barrier known as the blood-brainbarrier (BBB). The brain capillary endothelial cells (BCECs) that formthe BBB play important role in brain physiology by maintaining selectivepermeability and preventing passage of various compounds from the bloodinto the brain. One consequence of the highly effective barrierproperties of the BBB is the limited penetration of therapeutic agentsinto the brain, which makes treatment of many brain diseases extremelychallenging.

The persistence of the HIV virus in the CNS after HAART is thus,believed to be due to the inability of many of the HAART drugs to crossthe blood brain barrier (BBB) (Varatharajan et al, 2009). To assesspenetration of the HAART drugs into the brain, a scoring system has beendeveloped, known as the CNS penetration effectiveness (CPE) index, whichranks antiretroviral drugs' ability to enter the brain [Letendre et al,2008].

Efforts to improve the permeation of HIV drugs across the BBB have beenattempted, but have not proven therapeutically successful. For example,the oldest antiretroviral drug, AZT, was encapsulated in a magnetizedliposome to help it cross the BBB, but that delivery system did notprovide sufficient improvement in bioavailability to be usedtherapeutically [Saiyed et al, 2010].

Some of the most widely used and effective (in terms of reducing HIVload in the circulation) antiretroviral drugs have the lowest (worst)CPE rating. For example, tenofovir, a component of the combination pillsTruvad and Atripla, is widely used in HAART, but it hardly crosses theBBB, if at all (CPE rating of 1).

Efforts to improve the permeation of biologically active compoundsacross the BBB using amphiphilic vesicles have been attempted.

For example, complexation of the anionic carboxyfluorescein (CF) (afluorescent marker) with single headed amphiphiles of opposite charge incationic vesicles, formed by mixing single-tailed cationic and anionicsurfactants has been reported (Danoff et al. 2007). In addition tocomplexation a certain portion of the CF is passively encapsulatedwithin the core of the formed vesicles. And our present invention usingbolaamphiphiles includes the embodiments wherein a portion of the activeagent may be complexed to the head groups of the bolaamphiphiles andanother fraction of the active agents are encapsulated within the coreof the vesicles. In many embodiments the major portion of the activeagent is encapsulated by complexation with the head groups.

Furthermore, WO 02/055011 and WO 03/047499, both of the same applicantof the present invention, disclose amphiphilic derivatives composed ofat least one fatty acid chain derived from natural vegetable oils suchas vernonia oil, lesquerella oil and castor oil, in which functionalgroups such as epoxy, hydroxy and double bonds were modified into polarand ionic headgroups.

Additionally, WO 10/128504 reports a series of amphiphiles andbolamphiphiles (amphiphiles with two head groups) useful for targeteddrug delivery of insulin, insulin analogs, TNF, GDNF, DNA, RNA(including siRNA), enkephalin class of analgesics, and others.

These synthetic bolaamphiphiles (bolas) have recently been shown to formnanovesicles that interact with and encapsulate a variety of small andlarge molecules including peptides, proteins and plasmid DNAs deliveringthem across biological membranes. These bolaamphiphiles are a uniqueclass of compounds that have two hydrophilic headgroups placed at eachends of a hydrophobic domain. Bolaamphiphiles can form vesicles thatconsist of monolayer membrane that surrounds an aqueous core. Vesiclesmade from natural bolaamphiphiles, such as those extracted fromarchaebacteria (archaesomes), are very stable and, therefore, might beemployed for targeted drug delivery. However, bolaamphiphiles fromarchaebacteria are heterogeneous and cannot be easily extracted orchemically synthesized.

There are however no efficient delivery systems for delivery ofeffective HIV drugs into the CNS after systemic administration. Thus,there remains a need to make new compositions and for novel methods todeliver HIV active drugs into the brain. The compounds, compositions,and methods described herein are directed toward this end.

SUMMARY OF THE INVENTION

In certain aspects, provided herein are pharmaceutical compositionscomprising of a bolaamphiphile complex.

In further aspects, provided herein are novel nano-sized vesiclescomprising of bolaamphiphilic compounds.

In certain aspects, provided herein are novel bolaamphiphile complexescomprising one or more bolaamphiphilic compounds and a compound activeagainst HIV.

In further aspects, provided herein are novel formulations of HIV activecompounds with one or more bolaamphiphilic compounds or withbolaamphiphile vesicles. In a further aspect there are submicronvesicles with a monolayer membrane or bilayer membrane encapsulating aninner core, with an HIV active compound encapsulated within its coreand/or complexed to the inner and our head groups of the bolaamphiphilewhich comprise the said membrane.

In another aspect, provided here are methods of delivering HIV activedrugs agents into animal or human brain. In one embodiment, the methodcomprises the step of administering to the animal or human apharmaceutical composition comprising of a bolaamphiphile complex; andwherein the bolaamphiphile complex comprises one or more bolaamphiphiliccompounds and a compound active against HIV. In one particularembodiment, the HIV active drug is Tenofovir or({[(2R)-1-(6-amino-9H-purin-9-yl)propan-2-yl]oxy}methyl)phosphonic acid.In another particular embodiment, the HIV active drug is fosamprenavir.In another particular embodiment, the HIV active drug is enfuvirtide. Inanother particular embodiment, the HIV active drug is saquinavir. Inanother particular embodiment, the HIV active drug is lamivudine. Inanother particular embodiment, the HIV active drug is stavudine. In oneimportant embodiment the said bolaamphiphiles of the saidbolaamphiphilic complex comprises an aggregate which is a submicronvesicles wherein the said bolaamphiphiles comprise the membrane of theaggregate which encapsulate an inner core.

In one embodiment, the bolaamphiphilic compound consists of twohydrophilic headgroups linked through a long hydrophobic chain. Inanother embodiment, the hydrophilic headgroup is an amino containinggroup. In a specific embodiment, the hydrophilic headgroup is a tertiaryor quaternary amino containing group.

In one particular embodiment, the bolaamphiphilic compound is a compoundaccording to formula I:

HG²-L¹-HG¹  I

or a pharmaceutically acceptable salt, solvate, hydrate, prodrug,stereoisomer, tautomer, isotopic variant, or N-oxide thereof, or acombination thereof;wherein:

each HG¹ and HG² is independently a hydrophilic head group; and

L¹ is alkylene, alkenyl, heteroalkylene, or heteroalkenyl linker;unsubstituted or substituted with C₁-C₂₀ alkyl, hydroxyl, or oxo.

In one embodiment, the pharmaceutically acceptable salt is a quaternaryammonium salt.

In one embodiment, with respect to the bolaamphiphilic compound offormula I, the bolaamphiphilic compound is a compound according toformula II, III, IV, V, or VI:

or a pharmaceutically acceptable salt, solvate, hydrate, prodrug,stereoisomer, tautomer, isotopic variant, or N-oxide thereof, or acombination thereof;wherein:

each HG¹ and HG² is independently a hydrophilic head group;

each Z¹ and Z² is independently —C(R³)₂—, —N(R³)— or —O—;

R^(1a), R^(1b), R³, and R⁴ is independently H or C₁-C₈ alkyl;

each R^(2a) and R^(2b) is independently H, C₁-C₈ alkyl, OH, alkoxy, orO-HG¹ or O-HG²;

each n8, n9, n11, and n12 is independently an integer from 1-20;

n10 is an integer from 2-20; and

each dotted bond is independently a single or a double bond.

In one embodiment, with respect to the bolaamphiphilic compound offormula I, II, III, IV, V, or VI, each HG¹ and HG² is independentlyselected from:

wherein:

-   -   X is —NR^(5a)R^(5b), or —N⁺R^(5a)R^(5b)R^(5c); each R^(5a), and        R^(5b) is independently H or substituted or unsubstituted C₁-C₂₀        alkyl or R^(5a) and R^(5b) may join together to form an N        containing substituted or unsubstituted heteroaryl, or        substituted or unsubstituted heterocyclyl;    -   each R^(5c) is independently substituted or unsubstituted C₁-C₂₀        alkyl; each R⁸ is independently H, substituted or unsubstituted        C₁-C₂₀ alkyl, alkoxy, or carboxy;    -   m1 is 0 or 1; and    -   each n13, n14, and n15 is independently an integer from 1-20.

In certain embodiments, vesicle formation is carried out in the presenceof an additive that increases encapsulation of a therapeutic agent. Inone aspect of this embodiment, the additive is stearyl amine. In anotheraspect, the therapeutic agent is an antiviral agent, while in a stillfurther aspect the antiviral agent is Tenofovir. In still another aspectthe vesicles formed from the bolaamphiphiles contain additives that helpto stabilize the vesicles, by stabilizing the vesicle's membranes, suchas but not limited to cholesterol derivcatives such as cholesterylhemisuccinate and cholesterol itself and combinations such ascholesteryl hemisuccinate and cholesterol. In another embodiment thevesicles comprise the bolaamphiphiles, vesicle membrane stabilizingadditives, stearyl amine, and tenofovir. In still another embodimentsthe vesicles in addition to these components have another additiveswhich decorates the outer vesicle membranes with groups or pendants thatenhance penetration though biological barriers such as the BBB. A nonelimiting example of such additives may be alkyl conjugates of chitosanor bolaamphiphiles where one of the head groups is chitosan.

In certain embodiments, the present disclosure is directed to a methodof treatment of a patient afflicted with a viral disease comprisingadministration of vesicles described herein. In one aspect of thisembodiment, the patient is a human AIDS patient in need of suchtreatment. In particular aspects of this embodiment, the vesicles areformed in the presence of an additive that increases encapsulation of atherapeutic agent. In one aspect of this embodiment, the additive isstearyl amine. In another aspect, the therapeutic agent is an antiviralagent, while in a still further aspect the antiviral agent is Tenofovir.In another aspect of this embodiment, the virus is HIV.

Other objects and advantages will become apparent to those skilled inthe art from a consideration of the ensuing detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A: TEM micrograph of vesicles from GLH-20 FIG. 1B sizedistribution of vesicles from GLH-20 determined by DLS.

FIG. 2A: Head group hydrolysis by AChE of GLH-19 (▴ symbols) and GLH-20(♦ symbols). FIG. 2B: release of CF from GLH-19 vesicles. FIG. 2C:Release of CF from GLH-20 vesicles.

FIG. 3A: CF accumulation in brain after i.v. injection of encapsulatedand non-encapsulated CF. Only GLH-20 vesicles allow accumulation of CFin the brain. FIG. 3B demonstrates that CS improves GLH-20 vesicles'penetration into the brain.

FIG. 4A: Analgesia after i.v. injection of enkephalin non-encapsulatedand encapsulated in vesicles. Analgesia (compared with morphine, whichwas used as a positive control) is obtained only when enkephalin isencapsulated in GLH-20 vesicles, the head groups of which are hydrolyzedby ChE. FIG. 4B demonstrate that the vesicles do not disrupt the BBBsince non-encapsulated enkephalin co-injected with empty vesicles(extravesicular enkephalin) did not cause analgesia. **Significantlydifferent from free leu-enkephalin (t-test, P<0.01). ***Significantlydifferent from free leu-enkephalin (t-test, P<0.001).

FIG. 5A: Fluorescence in mouse cerebral cortex after i.v. injection ofalbumin-FITC (non-encapsulated) FIG. 5B: Fluorescence in mouse cerebralcortex after i.v. injection of albumin-FITC encapsulated in GLH-20vesicles.

FIG. 6A: The effect of the stearyl amine on tenofovir encapsulationmeasured at a bola concentration of 15 mg/ml in vesicles not containingstearyl amine FIG. 6B: the effect of stearyl amine on tenofovirencapsulation measured at a bola concentration of 10 mg/ml in vesiclesnot containing stearyl amine FIG. 6C: the effect of stearyl amine ontenofovir encapsulation measured at a bola concentration of 15 mg/ml invescicles containing 2.5 mg/ml stearyl amine. FIG. 6D: the effect ofstearyl amine on tenofovir encapsulation measured at a bolaconcentration of 10 mg/ml in vesicles containing 2.5 mg/ml stearylamine. Tenofovir is dissolved in HEPES in the concentration of 5 mg/ml

FIG. 7: Accumulation of CF in the brain after injecting the dyeencapsulated in naked vesicles (vesicles w/o CS), or in vesicles thatcontain CS-vernolate conjugate, or in vesicles that contain GLH-55a(vesicles CS bola), or in vesicles that contain GLH-55b (vesicles CSlong bola). CF-encapsulated vesicles were injected i.v. into mice and 30min afterward animals were sacrificed, brain removed, homogenized,deproteinized by trichloroacetic acid and fluorescence was determined inthe supernatants obtained by centrifugation after adjusting the pH to7.0.

FIG. 8: Chromoatogram of tenofovir (Panel A), Chromatogram ofsupernatant obtained from a brain homogenate followed bydeproteinization with TCA (Panel B), Chromatogram of and supernatant asin B after SPE treatment (Panel C).

FIG. 9: Chromatogram of the supernatant containing 40 μg tenofovir afterSPE treatment.

FIG. 10: Chromatogram of different concentrations of tenofovir, 5 μg/ml(Panel A), 10 μg/ml (Panel B), and 20 μg/ml (Panel C), in supernatantobtained after deproteinization of brain homogenate.

FIG. 11: A calibration curve of tenofovir in supernatant obtained frombrain homogenate.

FIG. 12: Chromatogram of tenofovir spiked into brain homogenate at 6.25μg/ml (Panel A), at 12.5 μg/ml (Panel B), and at 25 μg/ml (Panel C).

FIG. 13: Calibration curves of Tenofovir spiked into the supernatantobtained. from brain homogenate after removal of the proteins by TCA (A)and into brain homogenate (B).

FIG. 14A: Chromatogram of brain extracts taken from a first animalinjected with tenofovir-loaded V-Smart™ vesicles; FIG. 14B: Chromatogramof brain extracts taken from a second animal injected withtenofovir-loaded V-Smart™ vesicles; and FIG. 14C: Chromatogram of brainextracts taken from a third animal injected with tenofovir-loadedV-Smart™ vesicles.

FIG. 15: Determination of tenofovir concentrations in the brain afterdelivery of the drug by vesicles of the disclosure: Determination oftenofovir concentration in the brain after delivery in V-Smart vesicles(Panel A); Fragmentation of 2-deoxyadenosine-5-monophosphate (Panel B);and tenofovir concentration in mouse brain after injection ofencapsulated and non-encapsulated (free) tenofovir (Panel C).

FIG. 16A: Product ion mass spectra of Tenofovir EIC 176,206,270+MS2(288). FIG. 16B: Product ion mass spectra of 2′-Deoxyadenosine5′-phosphate EIC 136+MS2(332) in positive ionization mode.

FIG. 17A: Calibration curve for tenofovir in blood. FIG. 17B:Calibration Curve for tenofovir in brain. Various concentrations oftenofovir and a fixed concentration of the internal standard were spikedinto blood samples (FIG. 17A), or into brain homogenates (FIG. 17B), andthe samples were processed for deproteinization as described in thetext. The MS responses were measured after adding these samples to LC-MSand were plotted as a function of the ratio between tenofovir and theinternal standard. The ratio of tenofovir to internal standard allowedus to calculate percent recovery of tenofovir. Each sample was measuredin duplicates and the result of each duplicate is shown in the graph(filled circles), The average value of each duplicate (empty circles)were used to plot the calibration curve. The concentration range of thespiked tenofovir was 100-1000 ng/mL for the blood and 50-400 ng/mL forthe brain.

FIG. 18: Tenofovir concentration in the blood of mice after injectingthem with tenofovir, which was either encapsulated in GLH-19 vesicles,or in vesicles made from a mixture of the bolas GLH-19 and GLH-20. Inthe latter case, animals were either pretreated with pyridostigmine(with pyrido) or did not receive the pyrido pretreatment (w/o pyrido).Another group of animals was injected with empty vesicles and then withfree tenofovir. Results are mean of 4 mice±SD.

FIG. 19: Tenofovir concentration in the brain of mice after injectingthem with tenofovir, which was either encapsulated in GLH-19 vesicles,or in vesicles made from a mixture of the bolas GLH-19 and GLH-20. Inthe latter case, animals were either pretreated with pyridostigmine(with pyrido) or did not receive the pyrido pretreatment (w/o pyriso).Another group of animals was injected with empty vesicles and then withfree tenofovir, but tenofovir was not detected in the brain of thisgroup of mice and therefore no bars were assigned for this group.Results are mean of 4 mice±SD.

FIG. 20: Tenofovir concentrations in the brain of mice after injectingthe mice with optimized V-Smart™ vesicles made in 1 mL HEPES from 10 mgGLH-19 and GLH-20 at the ratio of 2:1, 1 mg GLH-55a, 2.4 mg cholesterylhemisuccinate, 1.6 mg cholesterol, 2.5 mg stearyl amine and 5 mg/mLtenofovir. Another group of mice were injected with free tenofovir. Thetotal dose of tenofovir that was injected to the mice that receivedencapsulated tenofovir was 37.5 m/kg, out of which 7.5 mg/kg wasencapsulated tenofovir. The total dose of tenofovir that was injected tothe mice that received free tenofovir was 75 mg/kg. All animals werepretreated with 0.5 mg/kg pyrido. Results are mean of 5 mice±SD.

DEFINITIONS Chemical Definitions

Definitions of specific functional groups and chemical terms aredescribed in more detail below. The chemical elements are identified inaccordance with the Periodic Table of the Elements, CAS version,Handbook of Chemistry and Physics, 75^(th) Ed., inside cover, andspecific functional groups are generally defined as described therein.Additionally, general principles of organic chemistry, as well asspecific functional moieties and reactivity, are described in ThomasSorrell, Organic Chemistry, University Science Books, Sausalito, 1999;Smith and March, March's Advanced Organic Chemistry, 5^(th) Edition,John Wiley & Sons, Inc., New York, 2001; Larock, Comprehensive OrganicTransformations, VCH Publishers, Inc., New York, 1989; and Carruthers,Some Modern Methods of Organic Synthesis, 3^(rd) Edition, CambridgeUniversity Press, Cambridge, 1987.

Compounds described herein can comprise one or more asymmetric centers,and thus can exist in various isomeric forms, e.g., enantiomers and/ordiastereomers. For example, the compounds described herein can be in theform of an individual enantiomer, diastereomer or geometric isomer, orcan be in the form of a mixture of stereoisomers, including racemicmixtures and mixtures enriched in one or more stereoisomer. Isomers canbe isolated from mixtures by methods known to those skilled in the art,including chiral high pressure liquid chromatography (HPLC) and theformation and crystallization of chiral salts; or preferred isomers canbe prepared by asymmetric syntheses. See, for example, Jacques et al.,Enantiomers, Racemates and Resolutions (Wiley Interscience, New York,1981); Wilen et al., Tetrahedron 33:2725 (1977); Eliel, Stereochemistryof Carbon Compounds (McGraw-Hill, NY, 1962); and Wilen, Tables ofResolving Agents and Optical Resolutions p. 268 (E. L. Eliel, Ed., Univ.of Notre Dame Press, Notre Dame, Ind. 1972). The invention additionallyencompasses compounds described herein as individual isomerssubstantially free of other isomers, and alternatively, as mixtures ofvarious isomers.

When a range of values is listed, it is intended to encompass each valueand sub-range within the range. For example “C₁₋₆ alkyl” is intended toencompass, C₁, C₂, C₃, C₄, C₅, C₆, C₁₋₆, C₁₋₅, C₁₋₄, C₁₋₃, C₁₋₂, C₂₋₆,C₂₋₅, C₂₋₄, C₂₋₃, C₃₋₆, C₃₋₅, C₃₋₄, C₄₋₆, C₄₋₅, and C₅₋₆ alkyl.

The following terms are intended to have the meanings presentedtherewith below and are useful in understanding the description andintended scope of the present invention. When describing the invention,which may include compounds, pharmaceutical compositions containing suchcompounds and methods of using such compounds and compositions, thefollowing terms, if present, have the following meanings unlessotherwise indicated. It should also be understood that when describedherein any of the moieties defined forth below may be substituted with avariety of substituents, and that the respective definitions areintended to include such substituted moieties within their scope as setout below. Unless otherwise stated, the term “substituted” is to bedefined as set out below. It should be further understood that the terms“groups” and “radicals” can be considered interchangeable when usedherein. The articles “a” and “an” may be used herein to refer to one orto more than one (i.e. at least one) of the grammatical objects of thearticle. By way of example “an analogue” means one analogue or more thanone analogue.

“Alkyl” refers to a radical of a straight-chain or branched saturatedhydrocarbon group having from 1 to 20 carbon atoms (“C₁₋₂₀ alkyl”). Insome embodiments, an alkyl group has 1 to 12 carbon atoms (“C₁₋₁₂alkyl”). In some embodiments, an alkyl group has 1 to 10 carbon atoms(“C₁₋₁₀ alkyl”). In some embodiments, an alkyl group has 1 to 9 carbonatoms (“C₁₋₉ alkyl”). In some embodiments, an alkyl group has 1 to 8carbon atoms (“C₁₋₈ alkyl”). In some embodiments, an alkyl group has 1to 7 carbon atoms (“C₁₋₂ alkyl”). In some embodiments, an alkyl grouphas 1 to 6 carbon atoms (“C₁₋₆ alkyl”, also referred to herein as “loweralkyl”). In some embodiments, an alkyl group has 1 to 5 carbon atoms(“C₁₋₅ alkyl”). In some embodiments, an alkyl group has 1 to 4 carbonatoms (“C₁₋₄ alkyl”). In some embodiments, an alkyl group has 1 to 3carbon atoms (“C₁₋₃ alkyl”). In some embodiments, an alkyl group has 1to 2 carbon atoms (“C₁₋₂ alkyl”). In some embodiments, an alkyl grouphas 1 carbon atom (“C₁₋₁₂ alkyl”). In some embodiments, an alkyl grouphas 2 to 6 carbon atoms (“C₂₋₆ alkyl”). Examples of C₁₋₆ alkyl groupsinclude methyl (C₁), ethyl (C₂), n-propyl (C₃), isopropyl (C₃), n-butyl(C₄), tert-butyl (C₄), sec-butyl (C₄), iso-butyl (C₄), n-pentyl (C₅),3-pentanyl (C₅), amyl (C₅), neopentyl (C₅), 3-methyl-2-butanyl (C₅),tertiary amyl (C₅), and n-hexyl (C₆). Additional examples of alkylgroups include n-heptyl (C₂), n-octyl (C₈) and the like. Unlessotherwise specified, each instance of an alkyl group is independentlyoptionally substituted, i.e., unsubstituted (an “unsubstituted alkyl”)or substituted (a “substituted alkyl”) with one or more substituents;e.g., for instance from 1 to 5 substituents, 1 to 3 substituents, or 1substituent. In certain embodiments, the alkyl group is unsubstitutedC₁₋₁₀ alkyl (e.g., CH₃). In certain embodiments, the alkyl group issubstituted C₁₋₁₀ alkyl.

“Alkylene” refers to a substituted or unsubstituted alkyl group, asdefined above, wherein two hydrogens are removed to provide a divalentradical. Exemplary divalent alkylene groups include, but are not limitedto, methylene (—CH₂—), ethylene (—CH₂CH₂—), the propylene isomers (e.g.,—CH₂CH₂CH₂— and —CH(CH₃)CH₂—) and the like.

“Alkenyl” refers to a radical of a straight-chain or branchedhydrocarbon group having from 2 to 20 carbon atoms, one or morecarbon-carbon double bonds, and no triple bonds (“C₂₋₂₀ alkenyl”). Insome embodiments, an alkenyl group has 2 to 10 carbon atoms (“C₂₋₁₀alkenyl”). In some embodiments, an alkenyl group has 2 to 9 carbon atoms(“C₂₋₉ alkenyl”). In some embodiments, an alkenyl group has 2 to 8carbon atoms (“C₂₋₈ alkenyl”). In some embodiments, an alkenyl group has2 to 7 carbon atoms (“C₂₋₇ alkenyl”). In some embodiments, an alkenylgroup has 2 to 6 carbon atoms (“C₂₋₆ alkenyl”). In some embodiments, analkenyl group has 2 to 5 carbon atoms (“C₂₋₅ alkenyl”). In someembodiments, an alkenyl group has 2 to 4 carbon atoms (“C₂₋₄ alkenyl”).In some embodiments, an alkenyl group has 2 to 3 carbon atoms (“C₂₋₃alkenyl”). In some embodiments, an alkenyl group has 2 carbon atoms (“C₂alkenyl”). The one or more carbon-carbon double bonds can be internal(such as in 2-butenyl) or terminal (such as in 1-butenyl). Examples ofC₂₋₄ alkenyl groups include ethenyl (C₂), 1-propenyl (C₃), 2-propenyl(C₃), 1-butenyl (C₄), 2-butenyl (C₄), butadienyl (C₄), and the like.Examples of C₂₋₆ alkenyl groups include the aforementioned C₂₋₄ alkenylgroups as well as pentenyl (C₅), pentadienyl (C₅), hexenyl (C₆), and thelike. Additional examples of alkenyl include heptenyl (C₇), octenyl(C₈), octatrienyl (C₈), and the like. Unless otherwise specified, eachinstance of an alkenyl group is independently optionally substituted,i.e., unsubstituted (an “unsubstituted alkenyl”) or substituted (a“substituted alkenyl”) with one or more substituents e.g., for instancefrom 1 to 5 substituents, 1 to 3 substituents, or 1 substituent. Incertain embodiments, the alkenyl group is unsubstituted C₂₋₁₀ alkenyl.In certain embodiments, the alkenyl group is substituted C₂₋₁₀ alkenyl.

“Alkenylene” refers a substituted or unsubstituted alkenyl group, asdefined above, wherein two hydrogens are removed to provide a divalentradical. Exemplary divalent alkenylene groups include, but are notlimited to, ethenylene (—CH═CH—), propenylenes (e.g., —CH═CHCH₂— and—C(CH₃)═CH— and —CH═C(CH₃)—) and the like.

“Alkynyl” refers to a radical of a straight-chain or branchedhydrocarbon group having from 2 to 20 carbon atoms, one or morecarbon-carbon triple bonds, and optionally one or more double bonds(“C₂₋₂₀ alkynyl”). In some embodiments, an alkynyl group has 2 to 10carbon atoms (“C₂₋₁₀ alkynyl”). In some embodiments, an alkynyl grouphas 2 to 9 carbon atoms (“C₂₋₉ alkynyl”). In some embodiments, analkynyl group has 2 to 8 carbon atoms (“C₂₋₈ alkynyl”). In someembodiments, an alkynyl group has 2 to 7 carbon atoms (“C₂₋₇ alkynyl”).In some embodiments, an alkynyl group has 2 to 6 carbon atoms (“C₂₋₆alkynyl”). In some embodiments, an alkynyl group has 2 to 5 carbon atoms(“C₂₋₅ alkynyl”). In some embodiments, an alkynyl group has 2 to 4carbon atoms (“C₂₋₄ alkynyl”). In some embodiments, an alkynyl group has2 to 3 carbon atoms (“C₂₋₃ alkynyl”). In some embodiments, an alkynylgroup has 2 carbon atoms (“C₂ alkynyl”). The one or more carbon-carbontriple bonds can be internal (such as in 2-butynyl) or terminal (such asin 1-butynyl). Examples of C₂₋₄ alkynyl groups include, withoutlimitation, ethynyl (C₂), 1-propynyl (C₃), 2-propynyl (C₃), 1-butynyl(C₄), 2-butynyl (C₄), and the like. Examples of C₂₋₆ alkenyl groupsinclude the aforementioned C₂₋₄ alkynyl groups as well as pentynyl (C₅),hexynyl (C₆), and the like. Additional examples of alkynyl includeheptynyl (C₇), octynyl (C₈), and the like. Unless otherwise specified,each instance of an alkynyl group is independently optionallysubstituted, i.e., unsubstituted (an “unsubstituted alkynyl”) orsubstituted (a “substituted alkynyl”) with one or more substituents;e.g., for instance from 1 to 5 substituents, 1 to 3 substituents, or 1substituent. In certain embodiments, the alkynyl group is unsubstitutedC₂₋₁₀ alkynyl. In certain embodiments, the alkynyl group is substitutedC₂₋₁₀ alkynyl.

“Alkynylene” refers a substituted or unsubstituted alkynyl group, asdefined above, wherein two hydrogens are removed to provide a divalentradical. Exemplary divalent alkynylene groups include, but are notlimited to, ethynylene, propynylene, and the like.

“Aryl” refers to a radical of a monocyclic or polycyclic (e.g., bicyclicor tricyclic) 4n+2 aromatic ring system (e.g., having 6, 10, or 14 πelectrons shared in a cyclic array) having 6-14 ring carbon atoms andzero heteroatoms provided in the aromatic ring system (“C₆₋₁₄ aryl”). Insome embodiments, an aryl group has six ring carbon atoms (“C₆ aryl”;e.g., phenyl). In some embodiments, an aryl group has ten ring carbonatoms (“C₁₀ aryl”; e.g., naphthyl such as 1-naphthyl and 2-naphthyl). Insome embodiments, an aryl group has fourteen ring carbon atoms (“C₁₄aryl”; e.g., anthracyl). “Aryl” also includes ring systems wherein thearyl ring, as defined above, is fused with one or more carbocyclyl orheterocyclyl groups wherein the radical or point of attachment is on thearyl ring, and in such instances, the number of carbon atoms continue todesignate the number of carbon atoms in the aryl ring system. Typicalaryl groups include, but are not limited to, groups derived fromaceanthrylene, acenaphthylene, acephenanthrylene, anthracene, azulene,benzene, chrysene, coronene, fluoranthene, fluorene, hexacene,hexaphene, hexylene, as-indacene, s-indacene, indane, indene,naphthalene, octacene, octaphene, octalene, ovalene, penta-2,4-diene,pentacene, pentalene, pentaphene, perylene, phenalene, phenanthrene,picene, pleiadene, pyrene, pyranthrene, rubicene, triphenylene, andtrinaphthalene. Particularly aryl groups include phenyl, naphthyl,indenyl, and tetrahydronaphthyl. Unless otherwise specified, eachinstance of an aryl group is independently optionally substituted, i.e.,unsubstituted (an “unsubstituted aryl”) or substituted (a “substitutedaryl”) with one or more substituents. In certain embodiments, the arylgroup is unsubstituted C₆₋₁₄ aryl. In certain embodiments, the arylgroup is substituted C₆₋₁₄ aryl.

In certain embodiments, an aryl group substituted with one or more ofgroups selected from halo, C₁-C₈ alkyl, C₁-C₈ haloalkyl, cyano, hydroxy,C₁-C₈ alkoxy, and amino.

Examples of representative substituted aryls include the following

In these formulae one of R⁵⁶ and R⁵⁷ may be hydrogen and at least one ofR⁵⁶ and R⁵⁷ is each independently selected from C₁-C₈ alkyl, C₁-C₈haloalkyl, 4-10 membered heterocyclyl, alkanoyl, C₁-C₈ alkoxy,heteroaryloxy, alkylamino, arylamino, heteroarylamino, NR⁵⁸COR⁵⁹,NR⁵⁸SOR⁵⁹NR⁵⁸SO₂R⁵⁹, COOalkyl, COOaryl, CONR⁵⁸R⁵⁹, CONR⁵⁸OR⁵⁹, NR⁵⁸R⁵⁹,SO₂NR⁵⁸R⁵⁹, S-alkyl, SOalkyl, SO₂alkyl, Saryl, SOaryl, SO₂aryl; or R⁵⁶and R⁵⁷ may be joined to form a cyclic ring (saturated or unsaturated)from 5 to 8 atoms, optionally containing one or more heteroatomsselected from the group N, O, or S. R⁶⁰ and R⁶¹ are independentlyhydrogen, C₁-C₈ alkyl, C₁-C₄ haloalkyl, C₃-C₁₀ cycloalkyl, 4-10 memberedheterocyclyl, C₆-C₁₀ aryl, substituted C₆-C₁₀ aryl, 5-10 memberedheteroaryl, or substituted 5-10 membered heteroaryl.

“Fused aryl” refers to an aryl having two of its ring carbon in commonwith a second aryl ring or with an aliphatic ring.

“Aralkyl” is a subset of alkyl and aryl, as defined herein, and refersto an optionally substituted alkyl group substituted by an optionallysubstituted aryl group.

“Heteroaryl” refers to a radical of a 5-10 membered monocyclic orbicyclic 4n+2 aromatic ring system (e.g., having 6 or 10 π electronsshared in a cyclic array) having ring carbon atoms and 1-4 ringheteroatoms provided in the aromatic ring system, wherein eachheteroatom is independently selected from nitrogen, oxygen and sulfur(“5-10 membered heteroaryl”). In heteroaryl groups that contain one ormore nitrogen atoms, the point of attachment can be a carbon or nitrogenatom, as valency permits. Heteroaryl bicyclic ring systems can includeone or more heteroatoms in one or both rings. “Heteroaryl” includes ringsystems wherein the heteroaryl ring, as defined above, is fused with oneor more carbocyclyl or heterocyclyl groups wherein the point ofattachment is on the heteroaryl ring, and in such instances, the numberof ring members continue to designate the number of ring members in theheteroaryl ring system. “Heteroaryl” also includes ring systems whereinthe heteroaryl ring, as defined above, is fused with one or more arylgroups wherein the point of attachment is either on the aryl orheteroaryl ring, and in such instances, the number of ring membersdesignates the number of ring members in the fused (aryl/heteroaryl)ring system. Bicyclic heteroaryl groups wherein one ring does notcontain a heteroatom (e.g., indolyl, quinolinyl, carbazolyl, and thelike) the point of attachment can be on either ring, i.e., either thering bearing a heteroatom (e.g., 2-indolyl) or the ring that does notcontain a heteroatom (e.g., 5-indolyl).

In some embodiments, a heteroaryl group is a 5-10 membered aromatic ringsystem having ring carbon atoms and 1-4 ring heteroatoms provided in thearomatic ring system, wherein each heteroatom is independently selectedfrom nitrogen, oxygen, and sulfur (“5-10 membered heteroaryl”). In someembodiments, a heteroaryl group is a 5-8 membered aromatic ring systemhaving ring carbon atoms and 1-4 ring heteroatoms provided in thearomatic ring system, wherein each heteroatom is independently selectedfrom nitrogen, oxygen, and sulfur (“5-8 membered heteroaryl”). In someembodiments, a heteroaryl group is a 5-6 membered aromatic ring systemhaving ring carbon atoms and 1-4 ring heteroatoms provided in thearomatic ring system, wherein each heteroatom is independently selectedfrom nitrogen, oxygen, and sulfur (“5-6 membered heteroaryl”). In someembodiments, the 5-6 membered heteroaryl has 1-3 ring heteroatomsselected from nitrogen, oxygen, and sulfur. In some embodiments, the 5-6membered heteroaryl has 1-2 ring heteroatoms selected from nitrogen,oxygen, and sulfur. In some embodiments, the 5-6 membered heteroaryl has1 ring heteroatom selected from nitrogen, oxygen, and sulfur. Unlessotherwise specified, each instance of a heteroaryl group isindependently optionally substituted, i.e., unsubstituted (an“unsubstituted heteroaryl”) or substituted (a “substituted heteroaryl”)with one or more substituents. In certain embodiments, the heteroarylgroup is unsubstituted 5-14 membered heteroaryl. In certain embodiments,the heteroaryl group is substituted 5-14 membered heteroaryl.

Exemplary 5-membered heteroaryl groups containing one heteroatominclude, without limitation, pyrrolyl, furanyl and thiophenyl. Exemplary5-membered heteroaryl groups containing two heteroatoms include, withoutlimitation, imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, andisothiazolyl. Exemplary 5-membered heteroaryl groups containing threeheteroatoms include, without limitation, triazolyl, oxadiazolyl, andthiadiazolyl. Exemplary 5-membered heteroaryl groups containing fourheteroatoms include, without limitation, tetrazolyl. Exemplary6-membered heteroaryl groups containing one heteroatom include, withoutlimitation, pyridinyl. Exemplary 6-membered heteroaryl groups containingtwo heteroatoms include, without limitation, pyridazinyl, pyrimidinyl,and pyrazinyl. Exemplary 6-membered heteroaryl groups containing threeor four heteroatoms include, without limitation, triazinyl andtetrazinyl, respectively. Exemplary 7-membered heteroaryl groupscontaining one heteroatom include, without limitation, azepinyl,oxepinyl, and thiepinyl. Exemplary 5,6-bicyclic heteroaryl groupsinclude, without limitation, indolyl, isoindolyl, indazolyl,benzotriazolyl, benzothiophenyl, isobenzothiophenyl, benzofuranyl,benzoisofuranyl, benzimidazolyl, benzoxazolyl, benzisoxazolyl,benzoxadiazolyl, benzthiazolyl, benzisothiazolyl, benzthiadiazolyl,indolizinyl, and purinyl. Exemplary 6,6-bicyclic heteroaryl groupsinclude, without limitation, naphthyridinyl, pteridinyl, quinolinyl,isoquinolinyl, cinnolinyl, quinoxalinyl, phthalazinyl, and quinazolinyl.

Examples of representative heteroaryls include the following:

wherein each Y is selected from carbonyl, N, NR⁶⁵, O, and S; and R⁶⁵ isindependently hydrogen, C₁-C₈ alkyl, C₃-C₁₀ cycloalkyl, 4-10 memberedheterocyclyl, C₆-C₁₀ aryl, and 5-10 membered heteroaryl.

Examples of representative aryl having hetero atoms containingsubstitution include the following:

wherein each W is selected from C(R⁶⁶)₂, NR⁶⁶, O, and S; and each Y isselected from carbonyl, NR⁶⁶, O and S; and R⁶⁶ is independentlyhydrogen, C₁-C₈ alkyl, C₃-C₁₀ cycloalkyl, 4-10 membered heterocyclyl,C₆-C₁₀ aryl, and 5-10 membered heteroaryl.

“Heteroaralkyl” is a subset of alkyl and heteroaryl, as defined herein,and refers to an optionally substituted alkyl group substituted by anoptionally substituted heteroaryl group.

“Carbocyclyl” or “carbocyclic” refers to a radical of a nonaromaticcyclic hydrocarbon group having from 3 to 10 ring carbon atoms (“C₃₋₁₀carbocyclyl”) and zero heteroatoms in the nonaromatic ring system. Insome embodiments, a carbocyclyl group has 3 to 8 ring carbon atoms(“C₃₋₈ carbocyclyl”). In some embodiments, a carbocyclyl group has 3 to6 ring carbon atoms (“C₃₋₆ carbocyclyl”). In some embodiments, acarbocyclyl group has 3 to 6 ring carbon atoms (“C₃₋₆ carbocyclyl”). Insome embodiments, a carbocyclyl group has 5 to 10 ring carbon atoms(“C₅₋₁₀ carbocyclyl”). Exemplary C₃₋₆ carbocyclyl groups include,without limitation, cyclopropyl (C₃), cyclopropenyl (C₃), cyclobutyl(C₄), cyclobutenyl (C₄), cyclopentyl (C₅), cyclopentenyl (C₅),cyclohexyl (C₆), cyclohexenyl (C₆), cyclohexadienyl (C₆), and the like.Exemplary C₃₋₈ carbocyclyl groups include, without limitation, theaforementioned C₃₋₆ carbocyclyl groups as well as cycloheptyl (C₇),cycloheptenyl (C₇), cycloheptadienyl (C₇), cycloheptatrienyl (C₇),cyclooctyl (C₈), cyclooctenyl (C₈), bicyclo[2.2.1]heptanyl (C₇),bicyclo[2.2.2]octanyl (C₈), and the like. Exemplary C₃₋₁₀ carbocyclylgroups include, without limitation, the aforementioned C₃₋₈ carbocyclylgroups as well as cyclononyl (C₉), cyclononenyl (C₉), cyclodecyl (C₁₀),cyclodecenyl (C₁₀), octahydro-1H-indenyl (C₉), decahydronaphthalenyl(C₁₀), spiro[4.5]decanyl (C₁₀), and the like. As the foregoing examplesillustrate, in certain embodiments, the carbocyclyl group is eithermonocyclic (“monocyclic carbocyclyl”) or contain a fused, bridged orspiro ring system such as a bicyclic system (“bicyclic carbocyclyl”) andcan be saturated or can be partially unsaturated. “Carbocyclyl” alsoincludes ring systems wherein the carbocyclyl ring, as defined above, isfused with one or more aryl or heteroaryl groups wherein the point ofattachment is on the carbocyclyl ring, and in such instances, the numberof carbons continue to designate the number of carbons in thecarbocyclic ring system. Unless otherwise specified, each instance of acarbocyclyl group is independently optionally substituted, i.e.,unsubstituted (an “unsubstituted carbocyclyl”) or substituted (a“substituted carbocyclyl”) with one or more substituents. In certainembodiments, the carbocyclyl group is unsubstituted C₃₋₁₀ carbocyclyl.In certain embodiments, the carbocyclyl group is a substituted C₃₋₁₀carbocyclyl.

In some embodiments, “carbocyclyl” is a monocyclic, saturatedcarbocyclyl group having from 3 to 10 ring carbon atoms (“C₃₋₁₀cycloalkyl”). In some embodiments, a cycloalkyl group has 3 to 8 ringcarbon atoms (“C₃₋₈ cycloalkyl”). In some embodiments, a cycloalkylgroup has 3 to 6 ring carbon atoms (“C₃₋₆ cycloalkyl”). In someembodiments, a cycloalkyl group has 5 to 6 ring carbon atoms (“C₅₋₆cycloalkyl”). In some embodiments, a cycloalkyl group has 5 to 10 ringcarbon atoms (“C₅₋₁₀ cycloalkyl”). Examples of C₅₋₆ cycloalkyl groupsinclude cyclopentyl (C₅) and cyclohexyl (C₅). Examples of C₃₋₆cycloalkyl groups include the aforementioned C₅₋₆ cycloalkyl groups aswell as cyclopropyl (C₃) and cyclobutyl (C₄). Examples of C₃₋₈cycloalkyl groups include the aforementioned C₃₋₆ cycloalkyl groups aswell as cycloheptyl (C₇) and cyclooctyl (C₈). Unless otherwisespecified, each instance of a cycloalkyl group is independentlyunsubstituted (an “unsubstituted cycloalkyl”) or substituted (a“substituted cycloalkyl”) with one or more substituents. In certainembodiments, the cycloalkyl group is unsubstituted C₃₋₁₀ cycloalkyl. Incertain embodiments, the cycloalkyl group is substituted C₃₋₁₀cycloalkyl.

“Heterocyclyl” or “heterocyclic” refers to a radical of a 3 to10-membered non aromatic ring system having ring carbon atoms and 1 to 4ring heteroatoms, wherein each heteroatom is independently selected fromnitrogen, oxygen, sulfur, boron, phosphorus, and silicon (“3-10 memberedheterocyclyl”). In heterocyclyl groups that contain one or more nitrogenatoms, the point of attachment can be a carbon or nitrogen atom, asvalency permits. A heterocyclyl group can either be monocyclic(“monocyclic heterocyclyl”) or a fused, bridged or spiro ring systemsuch as a bicyclic system (“bicyclic heterocyclyl”), and can besaturated or can be partially unsaturated. Heterocyclyl bicyclic ringsystems can include one or more heteroatoms in one or both rings.“Heterocyclyl” also includes ring systems wherein the heterocyclyl ring,as defined above, is fused with one or more carbocyclyl groups whereinthe point of attachment is either on the carbocyclyl or heterocyclylring, or ring systems wherein the heterocyclyl ring, as defined above,is fused with one or more aryl or heteroaryl groups, wherein the pointof attachment is on the heterocyclyl ring, and in such instances, thenumber of ring members continue to designate the number of ring membersin the heterocyclyl ring system. Unless otherwise specified, eachinstance of heterocyclyl is independently optionally substituted, i.e.,unsubstituted (an “unsubstituted heterocyclyl”) or substituted (a“substituted heterocyclyl”) with one or more substituents. In certainembodiments, the heterocyclyl group is unsubstituted 3-10 memberedheterocyclyl. In certain embodiments, the heterocyclyl group issubstituted 3-10 membered heterocyclyl.

In some embodiments, a heterocyclyl group is a 5-10 membered nonaromaticring system having ring carbon atoms and 1-4 ring heteroatoms, whereineach heteroatom is independently selected from nitrogen, oxygen, sulfur,boron, phosphorus, and silicon (“5-10 membered heterocyclyl”). In someembodiments, a heterocyclyl group is a 5-8 membered non aromatic ringsystem having ring carbon atoms and 1-4 ring heteroatoms, wherein eachheteroatom is independently selected from nitrogen, oxygen, and sulfur(“5-8 membered heterocyclyl”). In some embodiments, a heterocyclyl groupis a 5-6 membered nonaromatic ring system having ring carbon atoms and1-4 ring heteroatoms, wherein each heteroatom is independently selectedfrom nitrogen, oxygen, and sulfur (“5-6 membered heterocyclyl”). In someembodiments, the 5-6 membered heterocyclyl has 1-3 ring heteroatomsselected from nitrogen, oxygen, and sulfur. In some embodiments, the 5-6membered heterocyclyl has 1-2 ring heteroatoms selected from nitrogen,oxygen, and sulfur. In some embodiments, the 5-6 membered heterocyclylhas one ring heteroatom selected from nitrogen, oxygen, and sulfur.

Exemplary 3-membered heterocyclyl groups containing one heteroatominclude, without limitation, azirdinyl, oxiranyl, thiorenyl. Exemplary4-membered heterocyclyl groups containing one heteroatom include,without limitation, azetidinyl, oxetanyl and thietanyl. Exemplary5-membered heterocyclyl groups containing one heteroatom include,without limitation, tetrahydrofuranyl, dihydrofuranyl,tetrahydrothiophenyl, dihydrothiophenyl, pyrrolidinyl, dihydropyrrolyland pyrrolyl-2,5-dione. Exemplary 5-membered heterocyclyl groupscontaining two heteroatoms include, without limitation, dioxolanyl,oxasulfuranyl, disulfuranyl, and oxazolidin-2-one. Exemplary 5-memberedheterocyclyl groups containing three heteroatoms include, withoutlimitation, triazolinyl, oxadiazolinyl, and thiadiazolinyl. Exemplary6-membered heterocyclyl groups containing one heteroatom include,without limitation, piperidinyl, tetrahydropyranyl, dihydropyridinyl,and thianyl. Exemplary 6-membered heterocyclyl groups containing twoheteroatoms include, without limitation, piperazinyl, morpholinyl,dithianyl, dioxanyl. Exemplary 6-membered heterocyclyl groups containingtwo heteroatoms include, without limitation, triazinanyl. Exemplary7-membered heterocyclyl groups containing one heteroatom include,without limitation, azepanyl, oxepanyl and thiepanyl. Exemplary8-membered heterocyclyl groups containing one heteroatom include,without limitation, azocanyl, oxecanyl and thiocanyl. Exemplary5-membered heterocyclyl groups fused to a C₆ aryl ring (also referred toherein as a 5,6-bicyclic heterocyclic ring) include, without limitation,indolinyl, isoindolinyl, dihydrobenzofuranyl, dihydrobenzothienyl,benzoxazolinonyl, and the like. Exemplary 6-membered heterocyclyl groupsfused to an aryl ring (also referred to herein as a 6,6-bicyclicheterocyclic ring) include, without limitation, tetrahydroquinolinyl,tetrahydroisoquinolinyl, and the like.

Particular examples of heterocyclyl groups are shown in the followingillustrative examples:

wherein each W is selected from CR⁶⁷, C(R⁶⁷)₂, NR⁶⁷, O, and S; and eachY is selected from NR⁶⁷, O, and S; and R⁶⁷ is independently hydrogen,C₁-C₈ alkyl, C₃-C₁₀ cycloalkyl, 4-10 membered heterocyclyl, C₆-C₁₀ aryl,5-10 membered heteroaryl. These heterocyclyl rings may be optionallysubstituted with one or more substituents selected from the groupconsisting of the group consisting of acyl, acylamino, acyloxy, alkoxy,alkoxycarbonyl, alkoxycarbonylamino, amino, substituted amino,aminocarbonyl (carbamoyl or amido), aminocarbonylamino, aminosulfonyl,sulfonylamino, aryl, aryloxy, azido, carboxyl, cyano, cycloalkyl,halogen, hydroxy, keto, nitro, thiol, —S-alkyl, —S-aryl, —S(O)-alkyl,—S(O)-aryl, —S(O)₂-alkyl, and —S(O)₂-aryl. Substituting groups includecarbonyl or thiocarbonyl which provide, for example, lactam and ureaderivatives.

“Hetero” when used to describe a compound or a group present on acompound means that one or more carbon atoms in the compound or grouphave been replaced by a nitrogen, oxygen, or sulfur heteroatom. Heteromay be applied to any of the hydrocarbyl groups described above such asalkyl, e.g., heteroalkyl, cycloalkyl, e.g., heterocyclyl, aryl, e.g.,heteroaryl, cycloalkenyl, e.g., cycloheteroalkenyl, and the like havingfrom 1 to 5, and particularly from 1 to 3 heteroatoms.

“Acyl” refers to a radical —C(O)R²⁰, where R²⁰ is hydrogen, substitutedor unsubstituted alkyl, substituted or unsubstituted alkenyl,substituted or unsubstituted alkynyl, substituted or unsubstitutedcarbocyclyl, substituted or unsubstituted heterocyclyl, substituted orunsubstituted aryl, or substituted or unsubstituted heteroaryl, asdefined herein. “Alkanoyl” is an acyl group wherein R²⁰ is a group otherthan hydrogen. Representative acyl groups include, but are not limitedto, formyl (—CHO), acetyl (—C(═O)CH₃), cyclohexylcarbonyl,cyclohexylmethylcarbonyl, benzoyl (—C(═O)Ph), benzylcarbonyl(—C(═O)CH₂Ph), —C(O)—C₁-C₈ alkyl, —C(O)—(CH₂)_(t)(C₆-C₁₀ aryl),—C(O)—(CH₂)_(t)(5-10 membered heteroaryl), —C(O)—(CH₂)_(t)(C₃-C₁₀cycloalkyl), and —C(O)—(CH₂)_(t)(4-10 membered heterocyclyl), wherein tis an integer from 0 to 4. In certain embodiments, R²¹ is C₁-C₈ alkyl,substituted with halo or hydroxy; or C₃-C₁₀ cycloalkyl, 4-10 memberedheterocyclyl, C₆-C₁₀ aryl, arylalkyl, 5-10 membered heteroaryl orheteroarylalkyl, each of which is substituted with unsubstituted C₁-C₄alkyl, halo, unsubstituted C₁-C₄ alkoxy, unsubstituted C₁-C₄ haloalkyl,unsubstituted C₁-C₄ hydroxyalkyl, or unsubstituted C₁-C₄ haloalkoxy orhydroxy.

“Acylamino” refers to a radical —NR²²C(O)R²³, where each instance of R²²and R23 is independently hydrogen, substituted or unsubstituted alkyl,substituted or unsubstituted alkenyl, substituted or unsubstitutedalkynyl, substituted or unsubstituted carbocyclyl, substituted orunsubstituted heterocyclyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted heteroaryl, as defined herein, or R²² is anamino protecting group. Exemplary “acylamino” groups include, but arenot limited to, formylamino, acetylamino, cyclohexylcarbonylamino,cyclohexylmethyl-carbonylamino, benzoylamino and benzylcarbonylamino.Particular exemplary “acylamino” groups are —NR²⁴C(O)—C₁-C₈ alkyl,—NR²⁴C(O)—(CH₂)_(t)(C₆-C₁₀ aryl), —NR²⁴C(O)—(CH₂)_(t)(5-10 memberedheteroaryl), —NR²⁴C(O)—(CH₂)_(t)(C₃-C₁₀ cycloalkyl), and—NR²⁴C(O)—(CH₂)_(t)(4-10 membered heterocyclyl), wherein t is an integerfrom 0 to 4, and each R²⁴ independently represents H or C₁-C₈ alkyl. Incertain embodiments, R²⁵ is H, C₁-C₈ alkyl, substituted with halo orhydroxy; C₃-C₁₀ cycloalkyl, 4-10 membered heterocyclyl, C₆-C₁₀ aryl,arylalkyl, 5-10 membered heteroaryl or heteroarylalkyl, each of which issubstituted with unsubstituted C₁-C₄ alkyl, halo, unsubstituted C₁-C₄alkoxy, unsubstituted C₁-C₄ haloalkyl, unsubstituted C₁-C₄ hydroxyalkyl,or unsubstituted C₁-C₄ haloalkoxy or hydroxy; and R²⁶ is H, C₁-C₈ alkyl,substituted with halo or hydroxy;

C₃-C₁₀ cycloalkyl, 4-10 membered heterocyclyl, C₆-C₁₀ aryl, arylalkyl,5-10 membered heteroaryl or heteroarylalkyl, each of which issubstituted with unsubstituted C₁-C₄ alkyl, halo, unsubstituted C₁-C₄alkoxy, unsubstituted C₁-C₄ haloalkyl, unsubstituted C₁-C₄ hydroxyalkyl,or unsubstituted C₁-C₄ haloalkoxy or hydroxyl; provided that at leastone of R²⁵ and R²⁶ is other than H.

“Acyloxy” refers to a radical —OC(O)R²⁷, where R²⁷ is hydrogen,substituted or unsubstituted alkyl, substituted or unsubstitutedalkenyl, substituted or unsubstituted alkynyl, substituted orunsubstituted carbocyclyl, substituted or unsubstituted heterocyclyl,substituted or unsubstituted aryl, or substituted or unsubstitutedheteroaryl, as defined herein. Representative examples include, but arenot limited to, formyl, acetyl, cyclohexylcarbonyl,cyclohexylmethylcarbonyl, benzoyl and benzylcarbonyl. In certainembodiments, R²⁸ is C₁-C₈ alkyl, substituted with halo or hydroxy;C₃-C₁₀ cycloalkyl, 4-10 membered heterocyclyl, C₆-C₁₀ aryl, arylalkyl,5-10 membered heteroaryl or heteroarylalkyl, each of which issubstituted with unsubstituted C₁-C₄ alkyl, halo, unsubstituted C₁-C₄alkoxy, unsubstituted C₁-C₄ haloalkyl, unsubstituted C₁-C₄ hydroxyalkyl,or unsubstituted C₁-C₄ haloalkoxy or hydroxy.

“Alkoxy” refers to the group —OR²⁹ where R²⁹ is substituted orunsubstituted alkyl, substituted or unsubstituted alkenyl, substitutedor unsubstituted alkynyl, substituted or unsubstituted carbocyclyl,substituted or unsubstituted heterocyclyl, substituted or unsubstitutedaryl, or substituted or unsubstituted heteroaryl. Particular alkoxygroups are methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy,tert-butoxy, sec-butoxy, n-pentoxy, n-hexoxy, and 1,2-dimethylbutoxy.Particular alkoxy groups are lower alkoxy, i.e. with between 1 and 6carbon atoms. Further particular alkoxy groups have between 1 and 4carbon atoms.

In certain embodiments, R²⁹ is a group that has 1 or more substituents,for instance, from 1 to 5 substituents, and particularly from 1 to 3substituents, in particular 1 substituent, selected from the groupconsisting of amino, substituted amino, C₆-C₁₀ aryl, aryloxy, carboxyl,cyano, C₃-C₁₀ cycloalkyl, 4-10 membered heterocyclyl, halogen, 5-10membered heteroaryl, hydroxyl, nitro, thioalkoxy, thioaryloxy, thiol,alkyl-S(O)—, aryl-S(O)—, alkyl-S(O)₂— and aryl-S(O)₂—. Exemplary‘substituted alkoxy’ groups include, but are not limited to,O—(CH₂)_(t)(C₆-C₁₀ aryl), —O—(CH₂)_(t)(5-10 membered heteroaryl),—O—(CH₂)_(t)(C₃-C₁₀ cycloalkyl), and —O—(CH₂)_(t)(4-10 memberedheterocyclyl), wherein t is an integer from 0 to 4 and any aryl,heteroaryl, cycloalkyl or heterocyclyl groups present, may themselves besubstituted by unsubstituted C₁-C₄ alkyl, halo, unsubstituted C₁-C₄alkoxy, unsubstituted C₁-C₄ haloalkyl, unsubstituted C₁-C₄ hydroxyalkyl,or unsubstituted C₁-C₄ haloalkoxy or hydroxy. Particular exemplary‘substituted alkoxy’ groups are —OCF₃, —OCH₂CF₃, —OCH₂Ph,—OCH₂-cyclopropyl, —OCH₂CH₂OH, and —OCH₂CH₂NMe₂.

“Amino” refers to the radical —NH₂.

“Substituted amino” refers to an amino group of the formula —N(R³⁸)₂wherein R³⁸ is hydrogen, substituted or unsubstituted alkyl, substitutedor unsubstituted alkenyl, substituted or unsubstituted alkynyl,substituted or unsubstituted carbocyclyl, substituted or unsubstitutedheterocyclyl, substituted or unsubstituted aryl, substituted orunsubstituted heteroaryl, or an amino protecting group, wherein at leastone of R³⁸ is not a hydrogen. In certain embodiments, each R³⁸ isindependently selected from: hydrogen, C₁-C₈ alkyl, C₃-C₈ alkenyl, C₃-C₈alkynyl, C₆-C₁₀ aryl, 5-10 membered heteroaryl, 4-10 memberedheterocyclyl, or C₃-C₁₀ cycloalkyl; or C₁-C₈ alkyl, substituted withhalo or hydroxy; C₃-C₈ alkenyl, substituted with halo or hydroxy; C₃-C₈alkynyl, substituted with halo or hydroxy, or —(CH₂)_(t)(C₆-C₁₀ aryl),—(CH₂)_(t)(5-10 membered heteroaryl), —(CH₂)_(t)(C₃-C₁₀ cycloalkyl), or—(CH₂)_(t)(4-10 membered heterocyclyl), wherein t is an integer between0 and 8, each of which is substituted by unsubstituted C₁-C₄ alkyl,halo, unsubstituted C₁-C₄ alkoxy, unsubstituted C₁-C₄ haloalkyl,unsubstituted C₁-C₄ hydroxyalkyl, or unsubstituted C₁-C₄ haloalkoxy orhydroxy; or both R³⁸ groups are joined to form an alkylene group.

Exemplary ‘substituted amino’ groups are —NR³⁹—C₁-C₈ alkyl,—NR³⁹—(CH₂)_(t)(C₆-C₁₀ aryl), —NR³⁹—(CH₂)_(t)(5-10 membered heteroaryl),—NR³⁹—(CH₂)_(t)(C₃-C₁₀ cycloalkyl), and —NR³⁹—(CH₂)_(t)(4-10 memberedheterocyclyl), wherein t is an integer from 0 to 4, for instance 1 or 2,each R³⁹ independently represents H or C₁-C₈ alkyl; and any alkyl groupspresent, may themselves be substituted by halo, substituted orunsubstituted amino, or hydroxy; and any aryl, heteroaryl, cycloalkyl,or heterocyclyl groups present, may themselves be substituted byunsubstituted C₁-C₄ alkyl, halo, unsubstituted C₁-C₄ alkoxy,unsubstituted haloalkyl, unsubstituted C₁-C₄ hydroxyalkyl, orunsubstituted haloalkoxy or hydroxy. For the avoidance of doubt the term‘substituted amino’ includes the groups alkylamino, substitutedalkylamino, alkylarylamino, substituted alkylarylamino, arylamino,substituted arylamino, dialkylamino, and substituted dialkylamino asdefined below. Substituted amino encompasses both monosubstituted aminoand disubstituted amino groups.

“Azido” refers to the radical —N₃.

“Carbamoyl” or “amido” refers to the radical —C(O)NH₂.

“Substituted carbamoyl” or “substituted amido” refers to the radical—C(O)N(R⁶²)₂ wherein each R⁶² is independently hydrogen, substituted orunsubstituted alkyl, substituted or unsubstituted alkenyl, substitutedor unsubstituted alkynyl, substituted or unsubstituted carbocyclyl,substituted or unsubstituted heterocyclyl, substituted or unsubstitutedaryl, substituted or unsubstituted heteroaryl, or an amino protectinggroup, wherein at least one of R⁶² is not a hydrogen. In certainembodiments, R⁶² is selected from H, C₁-C₈ alkyl, C₃-C₁₀ cycloalkyl,4-10 membered heterocyclyl, C₆-C₁₀ aryl, aralkyl, 5-10 memberedheteroaryl, and heteroaralkyl; or C₁-C₈ alkyl substituted with halo orhydroxy; or C₃-C₁₀ cycloalkyl, 4-10 membered heterocyclyl, C₆-C₁₀ aryl,aralkyl, 5-10 membered heteroaryl, or heteroaralkyl, each of which issubstituted by unsubstituted C₁-C₄ alkyl, halo, unsubstituted C₁-C₄alkoxy, unsubstituted haloalkyl, unsubstituted C₁-C₄ hydroxyalkyl, orunsubstituted haloalkoxy or hydroxy; provided that at least one R⁶² isother than H.

Exemplary ‘substituted carbamoyl’ groups include, but are not limitedto, —C(O) NR⁶⁴—C₁-C₈ alkyl, —C(O)NR⁶⁴—(CH₂)_(t)(C₆-C₁₀ aryl),—C(O)N⁶⁴—(CH₂)_(t)(5-10 membered heteroaryl), —C(O)NR⁶⁴—(CH₂)_(t)(C₃-C₁₀cycloalkyl), and —C(O)NR⁶⁴—(CH₂)_(t)(4-10 membered heterocyclyl),wherein t is an integer from 0 to 4, each R⁶⁴ independently represents Hor C₁-C₈ alkyl and any aryl, heteroaryl, cycloalkyl or heterocyclylgroups present, may themselves be substituted by unsubstituted C₁-C₄alkyl, halo, unsubstituted C₁-C₄ alkoxy, unsubstituted haloalkyl,unsubstituted C₁-C₄ hydroxyalkyl, or unsubstituted haloalkoxy orhydroxy.

‘Carboxy’ refers to the radical —C(O)OH.

“Cyano” refers to the radical —CN.

“Halo” or “halogen” refers to fluoro (F), chloro (Cl), bromo (Br), andiodo (I). In certain embodiments, the halo group is either fluoro orchloro. In further embodiments, the halo group is iodo.

“Hydroxy” refers to the radical —OH.

“Nitro” refers to the radical —NO₂.

“Cycloalkylalkyl” refers to an alkyl radical in which the alkyl group issubstituted with a cycloalkyl group. Typical cycloalkylalkyl groupsinclude, but are not limited to, cyclopropylmethyl, cyclobutylmethyl,cyclopentylmethyl, cyclohexylmethyl, cycloheptylmethyl,cyclooctylmethyl, cyclopropylethyl, cyclobutylethyl, cyclopentylethyl,cyclohexylethyl, cycloheptylethyl, and cyclooctylethyl, and the like.

“Heterocyclylalkyl” refers to an alkyl radical in which the alkyl groupis substituted with a heterocyclyl group. Typical heterocyclylalkylgroups include, but are not limited to, pyrrolidinylmethyl,piperidinylmethyl, piperazinylmethyl, morpholinylmethyl,pyrrolidinylethyl, piperidinylethyl, piperazinylethyl, morpholinylethyl,and the like.

“Cycloalkenyl” refers to substituted or unsubstituted carbocyclyl grouphaving from 3 to 10 carbon atoms and having a single cyclic ring ormultiple condensed rings, including fused and bridged ring systems andhaving at least one and particularly from 1 to 2 sites of olefinicunsaturation. Such cycloalkenyl groups include, by way of example,single ring structures such as cyclohexenyl, cyclopentenyl,cyclopropenyl, and the like.

“Fused cycloalkenyl” refers to a cycloalkenyl having two of its ringcarbon atoms in common with a second aliphatic or aromatic ring andhaving its olefinic unsaturation located to impart aromaticity to thecycloalkenyl ring.

“Ethenyl” refers to substituted or unsubstituted —(C═C)—.

“Ethylene” refers to substituted or unsubstituted —(C—C)—.

“Ethynyl” refers to —(C≡C)—.

“Nitrogen-containing heterocyclyl” group means a 4- to 7-memberednon-aromatic cyclic group containing at least one nitrogen atom, forexample, but without limitation, morpholine, piperidine (e.g.2-piperidinyl, 3-piperidinyl and 4-piperidinyl), pyrrolidine (e.g.2-pyrrolidinyl and 3-pyrrolidinyl), azetidine, pyrrolidone, imidazoline,imidazolidinone, 2-pyrazoline, pyrazolidine, piperazine, and N-alkylpiperazines such as N-methyl piperazine. Particular examples includeazetidine, piperidone and piperazone.

“Thioketo” refers to the group ═S.

Alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroarylgroups, as defined herein, are optionally substituted (e.g.,“substituted” or “unsubstituted” alkyl, “substituted” or “unsubstituted”alkenyl, “substituted” or “unsubstituted” alkynyl, “substituted” or“unsubstituted” carbocyclyl, “substituted” or “unsubstituted”heterocyclyl, “substituted” or “unsubstituted” aryl or “substituted” or“unsubstituted” heteroaryl group). In general, the term “substituted”,whether preceded by the term “optionally” or not, means that at leastone hydrogen present on a group (e.g., a carbon or nitrogen atom) isreplaced with a permissible substituent, e.g., a substituent which uponsubstitution results in a stable compound, e.g., a compound which doesnot spontaneously undergo transformation such as by rearrangement,cyclization, elimination, or other reaction. Unless otherwise indicated,a “substituted” group has a substituent at one or more substitutablepositions of the group, and when more than one position in any givenstructure is substituted, the substituent is either the same ordifferent at each position. The term “substituted” is contemplated toinclude substitution with all permissible substituents of organiccompounds, any of the substituents described herein that results in theformation of a stable compound. The present invention contemplates anyand all such combinations in order to arrive at a stable compound. Forpurposes of this invention, heteroatoms such as nitrogen may havehydrogen substituents and/or any suitable substituent as describedherein which satisfy the valencies of the heteroatoms and results in theformation of a stable moiety.

Exemplary carbon atom substituents include, but are not limited to,halogen, —CN, —NO₂, —N₃, —SO₂H, —SO₃H, —OH, —OR^(aa), —ON(R^(bb))₂,—N(R^(bb))₂, —N(R^(bb))₃ ⁺X⁻, —N(OR^(cc))R^(bb), —SH, —SR^(aa),—SSR^(cc), —C(═O)R^(aa), —CO₂H, —CHO, —C(OR^(cc))₂, —CO₂R^(aa),—OC(═O)R^(aa), —OCO₂R^(aa), —C(═O)N(R^(bb))₂, —OC(═O)N(R^(bb))₂,—NR^(bb)C(═O)R^(aa), —NR^(bb)CO₂R^(aa), —NR^(bb)C(═O)N(R^(bb))₂,—C(═NR^(bb))R^(aa), —C(═NR^(bb))OR^(aa), —OC(═NR^(bb))R^(aa),—OC(═NR^(bb))OR^(aa), —C(═NR^(bb))N(R^(bb))₂, —OC(═NR^(bb))N(R^(bb))₂,—NR^(bb)C(═NR^(bb))N(R^(bb))₂, —C(═O)NR^(bb)SO₂R^(aa),—NR^(bb)SO₂R^(aa), —SO₂N(R^(bb))₂, —SO₂R^(aa), —SO₂OR^(aa), —OSO₂R^(aa),—S(═O)R^(aa), —OS(═O)R^(aa), —Si(R^(bb))₃,—OSi(R^(aa))₃—C(═S)N(R^(bb))₂, —C(═O)SR^(aa), —C(═S)SR^(aa),—SC(═S)SR^(aa), —SC(═O)SR^(aa), —OC(═O)SR^(aa), —SC(═O)OR^(aa),—SC(═O)R^(aa), —P(═O)₂R^(aa), —OP(═O)₂R^(aa), —P(═O)(R^(aa))₂,—OP(═O)(R^(aa))₂, —OP(═O)(OR^(cc))₂, —P(═O)₂N(R^(bb))₂,—OP(═O)₂N(R^(bb))₂, —P(═O)(NR^(bb))₂, —OP(═O)(NR^(bb))₂,—NR^(bb)P(═O)(OR^(cc))₂, —NR^(bb)P(═O)(NR^(bb))₂, —P(R^(cc))₂,—P(R^(cc))₃, —OP(R^(cc))₂, —OP(R^(cc))₃, —B(R^(aa))₂, —B(OR^(cc))₂,—BR^(aa)(OR^(cc)), C₁₋₁₀ alkyl, C₁₋₁₀ perhaloalkyl, C₂₋₁₀ alkenyl, C₂₋₁₀alkynyl, C₃₋₁₀ carbocyclyl, 3-14 membered heterocyclyl, C₆₋₁₄ aryl, and5-14 membered heteroaryl, wherein each alkyl, alkenyl, alkynyl,carbocyclyl, heterocyclyl, aryl, and heteroaryl is independentlysubstituted with 0, 1, 2, 3, 4, or 5 R^(dd) groups;

or two geminal hydrogens on a carbon atom are replaced with the group═O, ═S, ═NN(R^(bb))₂, ═NNR^(bb)C(═O)R^(aa), ═NNR^(bb)C(═O)OR^(aa),═NNR^(bb), or ═NOR^(cc); each instance of R^(aa) is, independently,selected from C₁₋₁₀ alkyl, C₁₋₁₀ perhaloalkyl, C₂₋₁₀ alkenyl, C₂₋₁₀alkynyl, C₃₋₁₀ carbocyclyl, 3-14 membered heterocyclyl, C₆₋₁₄ aryl, and5-14 membered heteroaryl, or two R^(aa) groups are joined to form a 3-14membered heterocyclyl or 5-14 membered heteroaryl ring, wherein eachalkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroarylis independently substituted with 0, 1, 2, 3, 4, or 5 R^(dd) groups;each instance of R^(bb) is, independently, selected from hydrogen, —OH,—OR^(aa), —N(R^(cc))₂, —CN, —C(═O)R^(aa), —C(═O)N(R^(cc))₂, —CO₂R^(aa),—SO₂R^(aa), —C(═NR^(cc))OR^(aa), —C(═NR^(cc))N(R^(cc))₂, —SO₂N(R^(cc))₂,—SO₂R^(cc), —SO₂OR^(cc), —SOR^(aa), —C(═S)N(R^(cc))₂, —C(═O)SR^(cc),—C(═S)SR^(cc), —P(═O)₂R^(aa), —P(═O)(R^(aa))₂, —P(═O)₂N(R^(cc))₂,—P(═O)(NR^(cc))₂, C₁₋₁₀ alkyl, C₁₋₁₀ perhaloalkyl, C₂₋₁₀ alkenyl, C₂₋₁₀alkynyl, C₃₋₁₀ carbocyclyl, 3-14 membered heterocyclyl, C₆₋₁₄ aryl, and5-14 membered heteroaryl, or two R^(bb) groups are joined to form a 3-14membered heterocyclyl or 5-14 membered heteroaryl ring, wherein eachalkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroarylis independently substituted with 0, 1, 2, 3, 4, or 5 R^(dd) groups;each instance of R^(cc) is, independently, selected from hydrogen, C₁₋₁₀alkyl, C₁₋₁₀ perhaloalkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, C₃₋₁₀carbocyclyl, 3-14 membered heterocyclyl, C₆₋₁₄ aryl, and 5-14 memberedheteroaryl, or two R^(cc) groups are joined to form a 3-14 memberedheterocyclyl or 5-14 membered heteroaryl ring, wherein each alkyl,alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl isindependently substituted with 0, 1, 2, 3, 4, or 5 R^(dd) groups;each instance of R^(dd) is, independently, selected from halogen, —CN,—NO₂, —N₃, —SO₂H, —SO₃H, —OH, —OR^(ee), —ON(R^(ff))₂, —N(R^(ff))₂,—N(R^(ff))₃ ⁺X⁻, —N(OR^(ee))R^(ff), —SH, —SR^(ee), —SSR^(ee),—C(═O)R^(ee), —CO₂H, —CO₂R^(ee), —OC(═O)R^(ee), —OCO₂R^(ee),—C(═O)N(R^(ff))₂, —OC(═O)N(R^(ff))₂, —NR^(ff)C(═O)R^(ee),—NR^(ff)CO₂R^(ee), —NR^(ff)C(═O)N(R^(ff))₂, —C(═NR^(ff))OR^(ee),—OC(═NR^(ff))R^(ee), —OC(═NR^(ff))OR^(ee), —C(═NR^(ff))N(R^(ff))₂,—OC(═NR^(ff))N(R^(ff))₂, —NR^(ff)C(═NR^(ff))N(R^(ff))₂,—NR^(ff)SO₂R^(ee), —SO₂N(R^(ff))₂, —SO₂R^(ee), —SO₂OR^(ee), —OSO₂R^(ee),—S(═O)R^(ee), —Si(R^(ee))₃, —OSi(R^(ee))₃, —C(═S)N(R^(ff))₂,—C(═O)SR^(ee), —C(═S)SR^(ee), —SC(═S)SR^(ee), —P(═O)₂R^(ee),—P(═O)(R^(ee))₂, —OP(═O)(R^(ee))₂, —OP(═O)(OR^(ee))₂, C₁₋₆ alkyl, C₁₋₆perhaloalkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₃₋₁₀ carbocyclyl, 3-10membered heterocyclyl, C₆₋₁₀ aryl, 5-10 membered heteroaryl, whereineach alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, andheteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 R^(gg)groups, or two geminal R^(dd) substituents can be joined to form ═O or═S;each instance of R^(ee) is, independently, selected from C₁₋₆ alkyl,C₁₋₆ perhaloalkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₃₋₁₀ carbocyclyl, C₆₋₁₀aryl, 3-10 membered heterocyclyl, and 3-10 membered heteroaryl, whereineach alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, andheteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 R^(gg)groups;each instance of R^(ff) is, independently, selected from hydrogen, C₁₋₆alkyl, C₁₋₆ perhaloalkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₃₋₁₀ carbocyclyl,3-10 membered heterocyclyl, C₆₋₁₀ aryl and 5-10 membered heteroaryl, ortwo R groups are joined to form a 3-14 membered heterocyclyl or 5-14membered heteroaryl ring, wherein each alkyl, alkenyl, alkynyl,carbocyclyl, heterocyclyl, aryl, and heteroaryl is independentlysubstituted with 0, 1, 2, 3, 4, or 5 R^(gg) groups; andeach instance of R^(gg) is, independently, halogen, —CN, —NO₂, —N₃,—SO₂H, —SO₃H, —OH, —OC₁₋₆ alkyl, —ON(C₁₋₆ alkyl)₂, —N(C₁₋₆ alkyl)₂,—N(C₁₋₆ alkyl)₃ ⁺X⁻, —NH(C₁₋₆ alkyl)₂ ⁺X⁻, —NH₂(C₁₋₆ alkyl)⁺X⁻, —NH₃⁺X⁻, —N(OC₁₋₆ alkyl)(C₁₋₆ alkyl), —N(OH)(C₁₋₆ alkyl), —NH(OH), —SH,—SC₁₋₆ alkyl, —SS(C₁₋₆ alkyl), —C(═O)(C₁₋₆ alkyl), —CO₂H, —OC₂(C₁₋₆alkyl), —OC(═O)(C₁₋₆ alkyl), —OCO₂(C₁₋₆ alkyl), —C(═O)NH₂, —C(═O)N(C₁₋₆alkyl)₂, —OC(═O)NH(C₁₋₆ alkyl), —NHC(═O)(C₁₋₆ alkyl), —N(C₁₋₆alkyl)C(═O)(C₁₋₆ alkyl), —NHCO₂(C₁₋₆ alkyl), —NHC(═O)N(C₁₋₆ alkyl)₂,—NHC(═O)NH(C₁₋₆ alkyl), —NHC(═O)NH₂, —C(═NH)O(C₁₋₆ alkyl), —OC(═NH)(C₁₋₆alkyl), —OC(═NH)OC₁₋₆ alkyl, —C(═NH)N(C₁₋₆ alkyl)₂, —C(═NH)NH(C₁₋₆alkyl), —C(═NH)NH₂, —OC(═NH)N(C₁₋₆ alkyl)₂, —OC(NH)NH(C₁₋₆ alkyl),—OC(NH)NH₂, —NHC(NH)N(C₁₋₆ alkyl)₂, —NHC(═NH)NH₂, —NHSO₂(C₁₋₆ alkyl),—SO₂N(C₁₋₆ alkyl)₂, —SO₂NH(C₁₋₆ alkyl), —SO₂NH₂, —SO₂C₁₋₆ alkyl,—SO₂OC₁₋₆ alkyl, —OSO₂C₁₋₆ alkyl, —SOC₁₋₆ alkyl, —Si(C₁₋₆ alkyl)₃,—OSi(C₁₋₆ alkyl)₃—C(═S)N(C₁₋₆ alkyl)₂, C(═S)NH(C₁₋₆ alkyl), C(═S)NH₂,—C(═O)S(C₁₋₆ alkyl), —C(═S)SC₁₋₆ alkyl, —SC(═S)SC₁₋₆ alkyl, —P(═O)₂(C₁₋₆alkyl), —P(═O)(C₁₋₆ alkyl)₂, —OP(═O)(C₁₋₆ alkyl)₂, —OP(═O)(OC₁₋₆alkyl)₂, C₁₋₆ alkyl, C₁₋₆ perhaloalkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl,C₃₋₁₆ carbocyclyl, C₆₋₁₀ aryl, 3-10 membered heterocyclyl, 5-10 memberedheteroaryl; or two geminal R^(gg) substituents can be joined to form ═Oor ═S; wherein X⁻ is a counterion.

A “counterion” or “anionic counterion” is a negatively charged groupassociated with a cationic quaternary amino group in order to maintainelectronic neutrality. Exemplary counterions include halide ions (e.g.,F⁻, Cl⁻, Br⁻, I⁻), NO₃ ⁻, ClO₄ ⁻, OH⁻, H₂PO₄ ⁻, HSO₄ ⁻, sulfonate ions(e.g., methansulfonate, trifluoromethanesulfonate, p-toluenesulfonate,benzenesulfonate, 10-camphor sulfonate, naphthalene-2-sulfonate,naphthalene-1-sulfonic acid-5-sulfonate, ethan-1-sulfonicacid-2-sulfonate, and the like), and carboxylate ions (e.g., acetate,ethanoate, propanoate, benzoate, glycerate, lactate, tartrate,glycolate, and the like).

Nitrogen atoms can be substituted or unsubstituted as valency permits,and include primary, secondary, tertiary, and quaternary nitrogen atoms.Exemplary nitrogen atom substitutents include, but are not limited to,hydrogen, —OH, —OR^(aa), —N(R^(cc))₂, —CN, —C(═O)R^(aa),—C(═O)N(R^(cc))₂, —CO₂R^(aa), —SO₂R^(aa), —C(═NR^(bb))R^(aa),—C(═NR^(cc))OR^(aa), —C(═NR^(cc))N(R^(cc))₂, —SO₂N(R^(cc))₂, —SO₂R^(cc),—SO₂OR^(cc), —SOR^(aa), —C(═S)N(R^(cc))₂, —C(═O)SR^(cc), —C(═S)SR^(cc),—P(═O)₂R^(aa), —P(═O)(R^(aa))₂, —P(═O)₂N(R^(cc))₂, —P(═O)(NR^(cc))₂,C₁₋₁₀ alkyl, C₁₋₁₀ perhaloalkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, C₃₋₁₀carbocyclyl, 3-14 membered heterocyclyl, C₆₋₁₄ aryl, and 5-14 memberedheteroaryl, or two R^(cc) groups attached to a nitrogen atom are joinedto form a 3-14 membered heterocyclyl or 5-14 membered heteroaryl ring,wherein each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl,and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5R^(dd) groups, and wherein R^(aa), R^(bb), R^(cc) and R^(dd) are asdefined above.

In certain embodiments, the substituent present on a nitrogen atom is anitrogen protecting group (also referred to as an amino protectinggroup). Nitrogen protecting groups include, but are not limited to, —OH,—OR^(aa), —N(R^(cc))₂, —C(═O)R^(aa), —C(═O)N(R^(cc))₂, —CO₂R^(aa),—SO₂R^(aa), —C(═NR^(cc))R^(aa), —C(═NR^(cc))OR^(aa),—C(═NR^(cc))N(R^(cc))₂, —SO₂N(R^(cc))₂, —SO₂R^(cc), —SO₂OR^(cc),—SOR^(aa), —C(═S)N(R^(cc))₂, —C(═O)SR^(cc), —C(═S)SR^(cc), C₁₋₁₀ alkyl(e.g., aralkyl, heteroaralkyl), C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, C₃₋₁₀carbocyclyl, 3-14 membered heterocyclyl, C₆₋₁₄ aryl, and 5-14 memberedheteroaryl groups, wherein each alkyl, alkenyl, alkynyl, carbocyclyl,heterocyclyl, aralkyl, aryl, and heteroaryl is independently substitutedwith 0, 1, 2, 3, 4, or 5 R^(dd) groups, and wherein R^(aa), R^(bb),R^(cc) and R^(dd) are as defined herein. Nitrogen protecting groups arewell known in the art and include those described in detail inProtecting Groups in Organic Synthesis, T. W. Greene and P. G. M. Wuts,3^(rd) edition, John Wiley & Sons, 1999, incorporated herein byreference.

For example, nitrogen protecting groups such as amide groups (e.g.,—C(═O)R^(aa)) include, but are not limited to, formamide, acetamide,chloroacetamide, trichloroacetamide, trifluoroacetamide,phenylacetamide, 3-phenylpropanamide, picolinamide,3-pyridylcarboxamide, N-benzoylphenylalanyl derivative, benzamide,p-phenylbenzamide, o-nitophenylacetamide, o-nitrophenoxyacetamide,acetoacetamide, (N′-dithiobenzyloxyacylamino)acetamide,3-(p-hydroxyphenyl)propanamide, 3-(o-nitrophenyl)propanamide,2-methyl-2-(o-nitrophenoxy)propanamide,2-methyl-2-(o-phenylazophenoxy)propanamide, 4-chlorobutanamide,3-methyl-3-nitrobutanamide, o-nitrocinnamide, N-acetylmethioninederivative, o-nitrobenzamide and o-(benzoyloxymethyl)benzamide.

Nitrogen protecting groups such as carbamate groups (e.g.,—C(═O)OR^(aa)) include, but are not limited to, methyl carbamate, ethylcarbamante, 9-fluorenylmethyl carbamate (Fmoc),9-(2-sulfo)fluorenylmethyl carbamate, 9-(2,7-dibromo)fluoroenylmethylcarbamate,2,7-di-t-butyl-[9-(10,10-dioxo-10,10,10,10-tetrahydrothioxanthyl)]methylcarbamate (DBD-Tmoc), 4-methoxyphenacyl carbamate (Phenoc),2,2,2-trichloroethyl carbamate (Troc), 2-trimethylsilylethyl carbamate(Teoc), 2-phenylethyl carbamate (hZ), 1-(1-adamantyl)-1-methylethylcarbamate (Adpoc), 1,1-dimethyl-2-haloethyl carbamate,1,1-dimethyl-2,2-dibromoethyl carbamate (DBtBOC),1,1-dimethyl-2,2,2-trichloroethyl carbamate (TCBOC),1-methyl-1-(4-biphenylyl)ethyl carbamate (Bpoc),1-(3,5-di-t-butylphenyl)-1-methylethyl carbamate (t-Bumeoc), 2-(2′- and4′-pyridyl)ethyl carbamate (Pyoc), 2-(N,N-dicyclohexylcarboxamido)ethylcarbamate, t-butyl carbamate (BOC), 1-adamantyl carbamate (Adoc), vinylcarbamate (Voc), allyl carbamate (Alloc), 1 isopropylallyl carbamate(Ipaoc), cinnamyl carbamate (Coc), 4-nitrocinnamyl carbamate (Noc),8-quinolyl carbamate, N hydroxypiperidinyl carbamate, alkyldithiocarbamate, benzyl carbamate (Cbz), p-methoxybenzyl carbamate (Moz),p-nitrobenzyl carbamate, p-bromobenzyl carbamate, p-chlorobenzylcarbamate, 2,4-dichlorobenzyl carbamate, 4-methylsulfinylbenzylcarbamate (Msz), 9-anthrylmethyl carbamate, diphenylmethyl carbamate,2-methylthioethyl carbamate, 2-methylsulfonylethyl carbamate,2-(p-toluenesulfonyl)ethyl carbamate, [2-(1,3-dithianyl)]methylcarbamate (Dmoc), 4-methylthiophenyl carbamate (Mtpc),2,4-dimethylthiophenyl carbamate (Bmpc), 2-phosphonioethyl carbamate(Peoc), 2-triphenylphosphonioisopropyl carbamate (Ppoc),1,1-dimethyl-2-cyanoethyl carbamate, mchloropacyloxybenzyl carbamate,p-(dihydroxyboryl)benzyl carbamate, 5-benzisoxazolylmethyl carbamate,2-(trifluoromethyl)-6-chromonylmethyl carbamate (Tcroc), m-nitrophenylcarbamate, 3,5-dimethoxybenzyl carbamate, o-nitrobenzyl carbamate,3,4-dimethoxy-6-nitrobenzyl carbamate, phenyl(o-nitrophenyl)methylcarbamate, t-amyl carbamate, S-benzyl thiocarbamate, p-cyanobenzylcarbamate, cyclobutyl carbamate, cyclohexyl carbamate, cyclopentylcarbamate, cyclopropylmethyl carbamate, p-decyloxybenzyl carbamate,2,2-dimethoxyacylvinyl carbamate, o-(N,N-dimethylcarboxamido)benzylcarbamate, 1,1-dimethyl-3-(N,N-dimethylcarboxamido)propyl carbamate,1,1-dimethylpropynyl carbamate, di(2-pyridyl)methyl carbamate,2-furanylmethyl carbamate, 2-iodoethyl carbamate, isoborynl carbamate,isobutyl carbamate, isonicotinyl carbamate,p-(p′-methoxyphenylazo)benzyl carbamate, 1-methylcyclobutyl carbamate,1-methylcyclohexyl carbamate, 1-methyl-1-cyclopropylmethyl carbamate,1-methyl-1-(3,5-dimethoxyphenyl)ethyl carbamate,1-methyl-1-(p-phenylazophenyl)ethyl carbamate, 1-methyl-1-phenylethylcarbamate, 1-methyl-1-(4-pyridyl)ethyl carbamate, phenyl carbamate,p-(phenylazo)benzyl carbamate, 2,4,6-tri-t-butylphenyl carbamate,4-(trimethylammonium)benzyl carbamate, and 2,4,6-trimethylbenzylcarbamate.

Nitrogen protecting groups such as sulfonamide groups (e.g.,—S(═O)₂R^(aa)) include, but are not limited to, p-toluenesulfonamide(Ts), benzenesulfonamide, 2,3,6,-trimethyl-4-methoxybenzenesulfonamide(Mtr), 2,4,6-trimethoxybenzenesulfonamide (Mtb),2,6-dimethyl-4-methoxybenzenesulfonamide (Pme),2,3,5,6-tetramethyl-4-methoxybenzenesulfonamide (Mte),4-methoxybenzenesulfonamide (Mbs), 2,4,6-trimethylbenzenesulfonamide(Mts), 2,6-dimethoxy-4-methylbenzenesulfonamide (iMds),2,2,5,7,8-pentamethylchroman-6-sulfonamide (Pmc), methanesulfonamide(Ms), β-trimethylsilylethanesulfonamide (SES), 9-anthracenesulfonamide,4-(4′,8′-dimethoxynaphthylmethyl)benzenesulfonamide (DNMBS),benzylsulfonamide, trifluoromethylsulfonamide, and phenacylsulfonamide.

Other nitrogen protecting groups include, but are not limited to,phenothiazinyl (10)-acyl derivative, N′-p-toluenesulfonylaminoacylderivative, N′-phenylaminothioacyl derivative, N-benzoylphenylalanylderivative, N-acetylmethionine derivative,4,5-diphenyl-3-oxazolin-2-one, N-phthalimide, N-dithiasuccinimide (Dts),N-2,3-diphenylmaleimide, N-2,5-dimethylpyrrole,N-1,1,4,4-tetramethyldisilylazacyclopentane adduct (STABASE),5-substituted 1,3-dimethyl-1,3,5-triazacyclohexan-2-one, 5-substituted1,3-dibenzyl-1,3,5-triazacyclohexan-2-one, 1-substituted3,5-dinitro-4-pyridone, N-methylamine, N-allylamine,N-[2-(trimethylsilyl)ethoxy]methylamine (SEM), N-3-acetoxypropylamine,N-(1-isopropyl-4-nitro-2-oxo-3-pyroolin-3-yl)amine, quaternary ammoniumsalts, N-benzylamine, N-di(4-methoxyphenyl)methylamine,N-5-dibenzosuberylamine, N-triphenylmethylamine (Tr),N-[(4-methoxyphenyl)diphenylmethyl]amine (MMTr),N-9-phenylfluorenylamine (PhF),N-2,7-dichloro-9-fluorenylmethyleneamine, N-ferrocenylmethylamino (Fcm),N-2-picolylamino N′-oxide, N-1,1-dimethylthiomethyleneamine,N-benzylideneamine, N-p-methoxybenzylideneamine,N-diphenylmethyleneamine, N-[(2-pyridyl)mesityl]methyleneamine,N—(N′,N′-dimethylaminomethylene)amine, N,N′-isopropylidenediamine,N-p-nitrobenzylideneamine, N-salicylideneamine,N-5-chlorosalicylideneamine,N-(5-chloro-2-hydroxyphenyl)phenylmethyleneamine,N-cyclohexylideneamine, N-(5,5-dimethyl-3-oxo-1-cyclohexenyl)amine,N-borane derivative, N-diphenylborinic acid derivative,N-[phenyl(pentaacylchromium- or tungsten)acyl]amine, N-copper chelate,N-zinc chelate, N-nitroamine, N-nitrosoamine, amine N-oxide,diphenylphosphinamide (Dpp), dimethylthiophosphinamide (Mpt),diphenylthiophosphinamide (Ppt), dialkyl phosphoramidates, dibenzylphosphoramidate, diphenyl phosphoramidate, benzenesulfenamide,o-nitrobenzenesulfenamide (Nps), 2,4-dinitrobenzenesulfenamide,pentachlorobenzenesulfenamide, 2-nitro-4-methoxybenzenesulfenamide,triphenylmethylsulfenamide, and 3-nitropyridinesulfenamide (Npys).

In certain embodiments, the substituent present on an oxygen atom is anoxygen protecting group (also referred to as a hydroxyl protectinggroup). Oxygen protecting groups include, but are not limited to,—R^(aa), —N(R^(bb))₂, —C(═O)SR^(aa), —C(═O)R^(aa), —CO₂R^(aa),—C(═O)N(R^(bb))₂, —C(═NR^(bb))R^(aa), —C(═NR^(bb))OR^(aa),—C(═NR^(bb))N(R^(bb))₂, —S(═O)R^(aa), —SO₂R^(aa), —Si(R^(aa))₃,—P(R^(cc))₂, —P(R^(cc))₃, —P(═O)₂R^(aa), —P(═O)(R^(aa))₂,—P(═O)(OR^(cc))₂, —P(═O)₂N(R^(bb))₂, and —P(═O)(NR^(bb))₂, whereinR^(aa), R^(bb), and R^(cc) are as defined herein. Oxygen protectinggroups are well known in the art and include those described in detailin Protecting Groups in Organic Synthesis, T. W. Greene and P. G. M.Wuts, 3^(rd) edition, John Wiley & Sons, 1999, incorporated herein byreference.

Exemplary oxygen protecting groups include, but are not limited to,methyl, methoxylmethyl (MOM), methylthiomethyl (MTM), t-butylthiomethyl,(phenyldimethylsilyl)methoxymethyl (SMOM), benzyloxymethyl (BOM),p-methoxybenzyloxymethyl (PMBM), (4-methoxyphenoxy)methyl (p-AOM),guaiacolmethyl (GUM), t-butoxymethyl, 4-pentenyloxymethyl (POM),siloxymethyl, 2-methoxyethoxymethyl (MEM), 2,2,2-trichloroethoxymethyl,bis(2-chloroethoxy)methyl, 2-(trimethylsilyl)ethoxymethyl (SEMOR),tetrahydropyranyl (THP), 3-bromotetrahydropyranyl,tetrahydrothiopyranyl, 1-methoxycyclohexyl, 4-methoxytetrahydropyranyl(MTHP), 4-methoxytetrahydrothiopyranyl, 4-methoxytetrahydrothiopyranylS,S-dioxide, 1-[(2-chloro-4-methyl)phenyl]-4-methoxypiperidin-4-yl(CTMP), 1,4-dioxan-2-yl, tetrahydrofuranyl, tetrahydrothiofuranyl,2,3,3a,4,5,6,7,7a-octahydro-7,8,8-trimethyl-4,7-methanobenzofuran-2-yl,1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, 1-methyl-1-methoxyethyl,1-methyl-1-benzyloxyethyl, 1-methyl-1-benzyloxy-2-fluoroethyl,2,2,2-trichloroethyl, 2-trimethylsilylethyl, 2-(phenylselenyl)ethyl,t-butyl, allyl, p-chlorophenyl, p-methoxyphenyl, 2,4-dinitrophenyl,benzyl (Bn), p-methoxybenzyl, 3,4-dimethoxybenzyl, o-nitrobenzyl,p-nitrobenzyl, p-halobenzyl, 2,6-dichlorobenzyl, p-cyanobenzyl,p-phenylbenzyl, 2-picolyl, 4-picolyl, 3-methyl-2-picolyl N-oxido,diphenylmethyl, p,p′-dinitrobenzhydryl, 5-dibenzosuberyl,triphenylmethyl, α-naphthyldiphenylmethyl,p-methoxyphenyldiphenylmethyl, di(p-methoxyphenyl)phenylmethyl,tri(p-methoxyphenyl)methyl, 4-(4′-bromophenacyloxyphenyl)diphenylmethyl,4,4′,4″-tris(4,5-dichlorophthalimidophenyl)methyl,4,4′,4″-tris(levulinoyloxyphenyl)methyl,4,4′,4″-tris(benzoyloxyphenyl)methyl,3-(imidazol-1-yl)bis(4′,4″-dimethoxyphenyl)methyl,1,1-bis(4-methoxyphenyl)-1′-pyrenylmethyl, 9-anthryl,9-(9-phenyl)xanthenyl, 9-(9-phenyl-10-oxo)anthryl,1,3-benzodisulfuran-2-yl, benzisothiazolyl S,S-dioxido, trimethylsilyl(TMS), triethylsilyl (TES), triisopropylsilyl (TIPS),dimethylisopropylsilyl (IPDMS), diethylisopropylsilyl (DEIPS),dimethylthexylsilyl, t-butyldimethylsilyl (TBDMS), t-butyldiphenylsilyl(TBDPS), tribenzylsilyl, tri-p-xylylsilyl, triphenylsilyl,diphenylmethylsilyl (DPMS), t-butylmethoxyphenylsilyl (TBMPS), formate,benzoylformate, acetate, chloroacetate, dichloroacetate,trichloroacetate, trifluoroacetate, methoxyacetate,triphenylmethoxyacetate, phenoxyacetate, pchlorophenoxyacetate,3-phenylpropionate, 4-oxopentanoate (levulinate),4,4-(ethylenedithio)pentanoate (levulinoyldithioacetal), pivaloate,adamantoate, crotonate, 4-methoxycrotonate, benzoate, p-phenylbenzoate,2,4,6-trimethylbenzoate (mesitoate), alkyl methyl carbonate,9-fluorenylmethyl carbonate (Fmoc), alkyl ethyl carbonate, alkyl2,2,2-trichloroethyl carbonate (Troc), 2-(trimethylsilyl)ethyl carbonate(TMSEC), 2-(phenylsulfonyl)ethyl carbonate (Psec),2-(triphenylphosphonio) ethyl carbonate (Peoc), alkyl isobutylcarbonate, alkyl vinyl carbonate alkyl allyl carbonate, alkylp-nitrophenyl carbonate, alkyl benzyl carbonate, alkyl p-methoxybenzylcarbonate, alkyl 3,4-dimethoxybenzyl carbonate, alkyl o-nitrobenzylcarbonate, alkyl p-nitrobenzyl carbonate, alkyl S-benzyl thiocarbonate,4-ethoxy-1-napththyl carbonate, methyl dithiocarbonate, 2-iodobenzoate,4-azidobutyrate, 4-nitro-4-methylpentanoate, o-(dibromomethyl)benzoate,2-formylbenzenesulfonate, 2-(methylthiomethoxy)ethyl,4-(methylthiomethoxy)butyrate, 2-(methylthiomethoxymethyl)benzoate,2,6-dichloro-4-methylphenoxyacetate,2,6-dichloro-4-(1,1,3,3-tetramethylbutyl)phenoxyacetate,2,4-bis(1,1-dimethylpropyl)phenoxyacetate, chlorodiphenylacetate,isobutyrate, monosuccinoate, (E)-2-methyl-2-butenoate,o-(methoxyacyl)benzoate, α-naphthoate, nitrate, alkylN,N,N′,N′-tetramethylphosphorodiamidate, alkyl N-phenylcarbamate,borate, dimethylphosphinothioyl, alkyl 2,4-dinitrophenylsulfenate,sulfate, methanesulfonate (mesylate), benzylsulfonate, and tosylate(Ts).

In certain embodiments, the substituent present on an sulfur atom is ansulfur protecting group (also referred to as a thiol protecting group).Sulfur protecting groups include, but are not limited to, —R^(aa),—N(R^(bb))₂, —C(═O)SR^(aa), —C(═O)R^(aa), —CO₂R^(aa), —C(═O)N(R^(bb))₂,—C(═NR^(bb))R^(aa), —C(═NR^(bb))OR^(aa), —C(═NR^(bb))N(R^(bb))₂,—S(═O)R^(aa), —SO₂R^(aa), —Si(R^(aa))₃, —P(R^(cc))₂, —P(R^(cc))₃,—P(═O)₂R^(aa), —P(═O)(R^(aa))₂, —P(═O)(OR^(cc))₂, —P(═O)₂N(R^(bb))₂, and—P(═O)(NR^(bb))₂, wherein R^(aa), R^(bb), and R^(cc) are as definedherein. Sulfur protecting groups are well known in the art and includethose described in detail in Protecting Groups in Organic Synthesis, T.W. Greene and P. G. M. Wuts, 3^(rd) edition, John Wiley & Sons, 1999,incorporated herein by reference.

“Compounds of the present invention”, and equivalent expressions, aremeant to embrace the compounds as hereinbefore described, in particularcompounds according to any of the Formula herein recited and/ordescribed, which expression includes the prodrugs, the pharmaceuticallyacceptable salts, and the solvates, e.g., hydrates, where the context sopermits. Similarly, reference to intermediates, whether or not theythemselves are claimed, is meant to embrace their salts, and solvates,where the context so permits.

These and other exemplary substituents are described in more detail inthe Detailed Description, Examples, and claims. The invention is notintended to be limited in any manner by the above exemplary listing ofsubstituents.

OTHER DEFINITIONS

“Pharmaceutically acceptable” means approved or approvable by aregulatory agency of the Federal or a state government or thecorresponding agency in countries other than the United States, or thatis listed in the U.S. Pharmacopoeia or other generally recognizedpharmacopoeia for use in animals, and more particularly, in humans.

“Pharmaceutically acceptable salt” refers to a salt of a compound of theinvention that is pharmaceutically acceptable and that possesses thedesired pharmacological activity of the parent compound. In particular,such salts are non-toxic may be inorganic or organic acid addition saltsand base addition salts. Specifically, such salts include: (1) acidaddition salts, formed with inorganic acids such as hydrochloric acid,hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and thelike; or formed with organic acids such as acetic acid, propionic acid,hexanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvic acid,lactic acid, malonic acid, succinic acid, malic acid, maleic acid,fumaric acid, tartaric acid, citric acid, benzoic acid,3-(4-hydroxybenzoyl) benzoic acid, cinnamic acid, mandelic acid,methanesulfonic acid, ethanesulfonic acid, 1,2-ethane-disulfonic acid,2-hydroxyethanesulfonic acid, benzenesulfonic acid,4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid,4-toluenesulfonic acid, camphorsulfonic acid,4-methylbicyclo[2.2.2]-oct-2-ene-1-carboxylic acid, glucoheptonic acid,3-phenylpropionic acid, trimethylacetic acid, tertiary butylacetic acid,lauryl sulfuric acid, gluconic acid, glutamic acid, hydroxynaphthoicacid, salicylic acid, stearic acid, muconic acid, and the like; or (2)salts formed when an acidic proton present in the parent compound eitheris replaced by a metal ion, e.g., an alkali metal ion, an alkaline earthion, or an aluminum ion; or coordinates with an organic base such asethanolamine, diethanolamine, triethanolamine, N-methylglucamine and thelike. Salts further include, by way of example only, sodium, potassium,calcium, magnesium, ammonium, tetraalkylammonium, and the like; and whenthe compound contains a basic functionality, salts of non toxic organicor inorganic acids, such as hydrochloride, hydrobromide, tartrate,mesylate, acetate, maleate, oxalate and the like. The term“pharmaceutically acceptable cation” refers to an acceptable cationiccounter-ion of an acidic functional group. Such cations are exemplifiedby sodium, potassium, calcium, magnesium, ammonium, tetraalkylammoniumcations, and the like (see, e.g., Berge, et al., J. Pharm. Sci. 66(1):1-79 (January'77).

“Pharmaceutically acceptable vehicle” refers to a diluent, adjuvant,excipient or carrier with which a compound of the invention isadministered.

“Pharmaceutically acceptable metabolically cleavable group” refers to agroup which is cleaved in vivo to yield the parent molecule of thestructural Formula indicated herein. Examples of metabolically cleavablegroups include —COR, —COOR, —CONRR and —CH₂OR radicals, where R isselected independently at each occurrence from alkyl, trialkylsilyl,carbocyclic aryl or carbocyclic aryl substituted with one or more ofalkyl, halogen, hydroxy or alkoxy. Specific examples of representativemetabolically cleavable groups include acetyl, methoxycarbonyl, benzoyl,methoxymethyl and trimethylsilyl groups.

“Prodrugs” refers to compounds, including derivatives of the compoundsof the invention, which have cleavable groups and become by solvolysisor under physiological conditions the compounds of the invention thatare pharmaceutically active in vivo. Such examples include, but are notlimited to, choline ester derivatives and the like, N-alkylmorpholineesters and the like. Other derivatives of the compounds of thisinvention have activity in both their acid and acid derivative forms,but in the acid sensitive form often offers advantages of solubility,tissue compatibility, or delayed release in the mammalian organism (see,Bundgard, H., Design of Prodrugs, pp. 7-9, 21-24, Elsevier, Amsterdam1985). Prodrugs include acid derivatives well know to practitioners ofthe art, such as, for example, esters prepared by reaction of the parentacid with a suitable alcohol, or amides prepared by reaction of theparent acid compound with a substituted or unsubstituted amine, or acidanhydrides, or mixed anhydrides. Simple aliphatic or aromatic esters,amides and anhydrides derived from acidic groups pendant on thecompounds of this invention are particular prodrugs. In some cases it isdesirable to prepare double ester type prodrugs such as (acyloxy)alkylesters or ((alkoxycarbonyl)oxy)alkylesters. Particularly the C₁ to C₈alkyl, C₂-C₈ alkenyl, C₂-C₈ alkynyl, aryl, C₂-C₁₂ substituted aryl, andC₂-C₁₂ arylalkyl esters of the compounds of the invention.

“Solvate” refers to forms of the compound that are associated with asolvent or water (also referred to as “hydrate”), usually by asolvolysis reaction. This physical association includes hydrogenbonding. Conventional solvents include water, ethanol, acetic acid andthe like. The compounds of the invention may be prepared e.g. incrystalline form and may be solvated or hydrated. Suitable solvatesinclude pharmaceutically acceptable solvates, such as hydrates, andfurther include both stoichiometric solvates and non-stoichiometricsolvates. In certain instances the solvate will be capable of isolation,for example when one or more solvent molecules are incorporated in thecrystal lattice of the crystalline solid. “Solvate” encompasses bothsolution-phase and isolable solvates. Representative solvates includehydrates, ethanolates and methanolates.

A “subject” to which administration is contemplated includes, but is notlimited to, humans (i.e., a male or female of any age group, e.g., apediatric subject (e.g, infant, child, adolescent) or adult subject(e.g., young adult, middleaged adult or senior adult)) and/or anon-human animal, e.g., a mammal such as primates (e.g., cynomolgusmonkeys, rhesus monkeys), cattle, pigs, horses, sheep, goats, rodents,cats, and/or dogs. In certain embodiments, the subject is a human. Incertain embodiments, the subject is a non-human animal. The terms“human”, “patient” and “subject” are used interchangeably herein.

“Therapeutically effective amount” means the amount of a compound that,when administered to a subject for treating a disease, is sufficient toeffect such treatment for the disease. The “therapeutically effectiveamount” can vary depending on the compound, the disease and itsseverity, and the age, weight, etc., of the subject to be treated.

“Preventing” or “prevention” refers to a reduction in risk of acquiringor developing a disease or disorder (i.e., causing at least one of theclinical symptoms of the disease not to develop in a subject not yetexposed to a disease-causing agent, or predisposed to the disease inadvance of disease onset.

The term “prophylaxis” is related to “prevention”, and refers to ameasure or procedure the purpose of which is to prevent, rather than totreat or cure a disease. Non-limiting examples of prophylactic measuresmay include the administration of vaccines; the administration of lowmolecular weight heparin to hospital patients at risk for thrombosisdue, for example, to immobilization; and the administration of ananti-malarial agent such as chloroquine, in advance of a visit to ageographical region where malaria is endemic or the risk of contractingmalaria is high.

“Treating” or “treatment” of any disease or disorder refers, in certainembodiments, to ameliorating the disease or disorder (i.e., arrestingthe disease or reducing the manifestation, extent or severity of atleast one of the clinical symptoms thereof). In another embodiment“treating” or “treatment” refers to ameliorating at least one physicalparameter, which may not be discernible by the subject. In yet anotherembodiment, “treating” or “treatment” refers to modulating the diseaseor disorder, either physically, (e.g., stabilization of a discerniblesymptom), physiologically, (e.g., stabilization of a physicalparameter), or both. In a further embodiment, “treating” or “treatment”relates to slowing the progression of the disease.

As used herein, the term “isotopic variant” refers to a compound thatcontains unnatural proportions of isotopes at one or more of the atomsthat constitute such compound. For example, an “isotopic variant” of acompound can contain one or more non-radioactive isotopes, such as forexample, deuterium (²H or D), carbon-13 (¹³C), nitrogen-15 (¹⁵N), or thelike. It will be understood that, in a compound where such isotopicsubstitution is made, the following atoms, where present, may vary, sothat for example, any hydrogen may be ²H/D, any carbon may be ¹³C, orany nitrogen may be ¹⁵N, and that the presence and placement of suchatoms may be determined within the skill of the art. Likewise, theinvention may include the preparation of isotopic variants withradioisotopes, in the instance for example, where the resultingcompounds may be used for drug and/or substrate tissue distributionstudies. The radioactive isotopes tritium, i.e., ³H, and carbon-14,i.e., ¹⁴C, are particularly useful for this purpose in view of theirease of incorporation and ready means of detection. Further, compoundsmay be prepared that are substituted with positron emitting isotopes,such as ¹¹C, ¹⁸F, ¹⁵O and ¹³N, and would be useful in Positron EmissionTopography (PET) studies for examining substrate receptor occupancy. Allisotopic variants of the compounds provided herein, radioactive or not,are intended to be encompassed within the scope of the invention.

It is also to be understood that compounds that have the same molecularformula but differ in the nature or sequence of bonding of their atomsor the arrangement of their atoms in space are termed “isomers”. Isomersthat differ in the arrangement of their atoms in space are termed“stereoisomers”.

Stereoisomers that are not mirror images of one another are termed“diastereomers” and those that are non-superimposable mirror images ofeach other are termed “enantiomers”. When a compound has an asymmetriccenter, for example, when it is bonded to four different groups, a pairof enantiomers is possible. An enantiomer can be characterized by theabsolute configuration of its asymmetric center and is described by theR- and S-sequencing rules of Cahn and Prelog, or by the manner in whichthe molecule rotates the plane of polarized light and designated asdextrorotatory or levorotatory (i.e., as (+) or (−)-isomersrespectively). A chiral compound can exist as either individualenantiomer or as a mixture thereof. A mixture containing equalproportions of the enantiomers is called a “racemic mixture”.

“Tautomers” refer to compounds that are interchangeable forms of aparticular compound structure, and that vary in the displacement ofhydrogen atoms and electrons. Thus, two structures may be in equilibriumthrough the movement of r electrons and an atom (usually H). Forexample, enols and ketones are tautomers because they are rapidlyinterconverted by treatment with either acid or base. Another example oftautomerism is the aci- and nitro-forms of phenylnitromethane, which arelikewise formed by treatment with acid or base. Tautomeric forms may berelevant to the attainment of the optimal chemical reactivity andbiological activity of a compound of interest.

As used herein a pure enantiomeric compound is substantially free fromother enantiomers or stereoisomers of the compound (i.e., inenantiomeric excess). In other words, an “S” form of the compound issubstantially free from the “R” form of the compound and is, thus, inenantiomeric excess of the “R” form. The term “enantiomerically pure” or“pure enantiomer” denotes that the compound comprises more than 75% byweight, more than 80% by weight, more than 85% by weight, more than 90%by weight, more than 91% by weight, more than 92% by weight, more than93% by weight, more than 94% by weight, more than 95% by weight, morethan 96% by weight, more than 97% by weight, more than 98% by weight,more than 98.5% by weight, more than 99% by weight, more than 99.2% byweight, more than 99.5% by weight, more than 99.6% by weight, more than99.7% by weight, more than 99.8% by weight or more than 99.9% by weight,of the enantiomer. In certain embodiments, the weights are based upontotal weight of all enantiomers or stereoisomers of the compound.

As used herein and unless otherwise indicated, the term“enantiomerically pure R-compound” refers to at least about 80% byweight R-compound and at most about 20% by weight S-compound, at leastabout 90% by weight R-compound and at most about 10% by weightS-compound, at least about 95% by weight R-compound and at most about 5%by weight S-compound, at least about 99% by weight R-compound and atmost about 1% by weight S-compound, at least about 99.9% by weightR-compound or at most about 0.1% by weight S-compound. In certainembodiments, the weights are based upon total weight of compound.

As used herein and unless otherwise indicated, the term“enantiomerically pure S-compound” or “S-compound” refers to at leastabout 80% by weight S-compound and at most about 20% by weightR-compound, at least about 90% by weight S-compound and at most about10% by weight R-compound, at least about 95% by weight S-compound and atmost about 5% by weight R-compound, at least about 99% by weightS-compound and at most about 1% by weight R-compound or at least about99.9% by weight S-compound and at most about 0.1% by weight R-compound.In certain embodiments, the weights are based upon total weight ofcompound.

In the compositions provided herein, an enantiomerically pure compoundor a pharmaceutically acceptable salt, solvate, hydrate or prodrugthereof can be present with other active or inactive ingredients. Forexample, a pharmaceutical composition comprising enantiomerically pureR-compound can comprise, for example, about 90% excipient and about 10%enantiomerically pure R-compound. In certain embodiments, theenantiomerically pure R-compound in such compositions can, for example,comprise, at least about 95% by weight R-compound and at most about 5%by weight S-compound, by total weight of the compound. For example, apharmaceutical composition comprising enantiomerically pure S-compoundcan comprise, for example, about 90% excipient and about 10%enantiomerically pure S-compound. In certain embodiments, theenantiomerically pure S-compound in such compositions can, for example,comprise, at least about 95% by weight S-compound and at most about 5%by weight R-compound, by total weight of the compound. In certainembodiments, the active ingredient can be formulated with little or noexcipient or carrier.

The compounds of this invention may possess one or more asymmetriccenters; such compounds can therefore be produced as individual (R)- or(S)-stereoisomers or as mixtures thereof.

Unless indicated otherwise, the description or naming of a particularcompound in the specification and claims is intended to include bothindividual enantiomers and mixtures, racemic or otherwise, thereof. Themethods for the determination of stereochemistry and the separation ofstereoisomers are well-known in the art.

One having ordinary skill in the art of organic synthesis will recognizethat the maximum number of heteroatoms in a stable, chemically feasibleheterocyclic ring, whether it is aromatic or non aromatic, is determinedby the size of the ring, the degree of unsaturation and the valence ofthe heteroatoms. In general, a heterocyclic ring may have one to fourheteroatoms so long as the heteroaromatic ring is chemically feasibleand stable.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

In certain aspects, provided herein are pharmaceutical compositionscomprising of a bolaamphiphile complex.

In further aspects, provided herein are novel nano-sized vesiclescomprising of bolaamphiphilic compounds.

In certain aspects, provided herein are novel bolaamphiphile complexescomprising one or more bolaamphiphilic compounds and a compound activeagainst HIV.

In further aspects, provided herein are novel formulations of HIV activecompounds with one or more bolaamphiphilic compounds or withbolaamphiphile vesicles.

In another aspect, provided here are methods of delivering HIV activedrugs agents into animal or human brain. In one embodiment, the methodcomprises the step of administering to the animal or human apharmaceutical composition comprising of a bolaamphiphile complex; andwherein the bolaamphiphile complex comprises one or more bolaamphiphiliccompounds and a compound active against HIV. In one particularembodiment, the HIV active drug is Tenofovir or({[(2R)-1-(6-amino-9H-purin-9-yl)propan-2-yl]oxy}methyl)phosphonic acid.In another particular embodiment, the HIV active drug is fosamprenavir.In another particular embodiment, the HIV active drug is enfuvirtide. Inanother particular embodiment, the HIV active drug is saquinavir. Inanother particular embodiment, the HIV active drug is lamivudine. Inanother particular embodiment, the HIV active drug is stavudine.

In one embodiment, the bolaamphiphilic complex comprises onebolaamphiphilic compound. In another embodiment, the bolaamphiphiliccomplex comprises two bolaamphiphilic compounds.

In one embodiment, the bolaamphiphilic compound consists of twohydrophilic headgroups linked through a long hydrophobic chain. Inanother embodiment, the hydrophilic headgroup is an amino containinggroup. In a specific embodiment, the hydrophilic headgroup is a tertiaryor quaternary amino containing group.

In one particular embodiment, the bolaamphiphilic compound is a compoundaccording to formula I:

HG²-L¹-HG¹  I

or a pharmaceutically acceptable salt, solvate, hydrate, prodrug,stereoisomer, tautomer, isotopic variant, or N-oxide thereof, or acombination thereof;wherein:

each HG¹ and HG² is independently a hydrophilic head group; and

L¹ is alkylene, alkenyl, heteroalkylene, or heteroalkenyl linker;unsubstituted or substituted with C₁-C₂₀ alkyl, hydroxyl, or oxo.

In one embodiment, the pharmaceutically acceptable salt is a quaternaryammonium salt.

In one embodiment, with respect to the bolaamphiphilic compound offormula I, L¹ is heteroalkylene, or heteroalkenyl linker comprising C,N, and O atoms; unsubstituted or substituted with C₁-C₂₀ alkyl,hydroxyl, or oxo.

In another embodiment, with respect to the bolaamphiphilic compound offormula I, L¹ is

—O-L²-C(O)—O—(CH₂)_(n4)—O—C(O)-L³-O—, or

—O-L²C(O)—O—(CH₂)_(n5)—O—C(O)—(CH₂)_(n6)—,

-   -   and wherein each L² and L³ is C₄-C₂₀ alkenyl linker;        unsubstituted or substituted with C₁-C₈ alkyl or hydroxy;    -   and n4, n5, and n6 is independently an integer from 4-20.

In one embodiment, each L² and L³ is independentlyC(R¹)—C(OH)—CH₂—(CH═CH)—(CH₂)_(n7)—; R¹ is C₁-C₈ alkyl, and n7 isindependently an integer from 4-20.

In another embodiment, with respect to the bolaamphiphilic compound offormula I, L¹ is —O—(CH₂)_(n1)—O—C(O)—(CH₂)_(n2)—C(O)—O—(CH₂)_(n3)—O—.

In another embodiment, with respect to the bolaamphiphilic compound offormula I, L¹ is

wherein:

-   -   each Z¹ and Z² is independently —C(R³)₂—, —N(R³)— or —O—;    -   each R^(1a), R^(1b), R³, and R⁴ is independently H or C₁-C₈        alkyl;    -   each R^(2a) and R^(2b) is independently H, C₁-C₈ alkyl, OH, or        alkoxy;    -   each n8, n9, n11, and n12 is independently an integer from 1-20;    -   n10 is an integer from 2-20; and    -   each dotted bond is independently a single or a double bond.    -   and wherein each methylene carbon is unsubstituted or        substituted with C₁-C₄ alkyl; and each n1, n2, and n3 is        independently an integer from 4-20.

In one embodiment, with respect to the bolaamphiphilic compound offormula I, the bolaamphiphilic compound is a compound according toformula II, III, IV, V, or VI:

or a pharmaceutically acceptable salt, solvate, hydrate, prodrug,stereoisomer, tautomer, isotopic variant, or N-oxide thereof, or acombination thereof;wherein:

each HG¹ and HG² is independently a hydrophilic head group;

each Z¹ and Z² is independently —C(R³)₂—, —N(R³)— or —O—;

each R^(1a), R^(1b), R³, and R⁴ is independently H or C₁-C₈ alkyl;

each R^(2a) and R^(2b) is independently H, C₁-C₈ alkyl, OH, alkoxy, orO-HG¹ or O-HG²;

each n8, n9, n11, and n12 is independently an integer from 1-20;

n10 is an integer from 2-20; and

each dotted bond is independently a single or a double bond.

In one embodiment, with respect to the bolaamphiphilic compound offormula II, III, IV, V, or VI, each n9 and n11 is independently aninteger from 2-12. In another embodiment, n9 and n11 is independently aninteger from 4-8. In a particular embodiment, each n9 and n11 is 7 or11.

In one embodiment, with respect to the bolaamphiphilic compound offormula II, III, IV, V, or VI, each n8 and n12 is independently 1, 2, 3,or 4. In a particular embodiment, each n8 and n12 is 1.

In one embodiment, with respect to the bolaamphiphilic compound offormula II, III, IV, V, or VI, each R^(2a) and R^(2b) is independentlyH, OH, or alkoxy. In another embodiment, each R^(2a) and R^(2b) isindependently H, OH, or OMe. In another embodiment, each R^(2a) andR^(2b) is independently-O-HG¹ or O-HG². In a particular embodiment, eachR^(2a) and R^(2b) is OH.

In one embodiment, with respect to the bolaamphiphilic compound offormula II, III, IV, V, or VI, each R^(1a) and R^(1b) is independentlyH, Me, Et, n-Pr, i-Pr, n-Bu, i-Bu, sec-Bu, n-pentyl, isopentyl, n-hexyl,n-heptyl, or n-octyl. In a particular embodiment, each R^(1a) and R^(1b)is independently n-pentyl.

In one embodiment, with respect to the bolaamphiphilic compound offormula II, III, IV, V, or VI, each dotted bond is a single bond. Inanother embodiment, each dotted bond is a double bond.

In one embodiment, with respect to the bolaamphiphilic compound offormula II, III, IV, V, or VI, n10 is an integer from 2-16. In anotherembodiment, n10 is an integer from 2-12. In a particular embodiment, n10is 2, 4, 6, 8, 10, 12, or 16.

In one embodiment, with respect to the bolaamphiphilic compound offormula IV, R⁴ is H, Me, Et, n-Pr, i-Pr, n-Bu, i-Bu, sec-Bu, n-pentyl,or isopentyl. In another embodiment, R⁴ is Me, or Et. In a particularembodiment, R⁴ is Me.

In one embodiment, with respect to the bolaamphiphilic compound offormula II, III, IV, V, or VI, each Z¹ and Z² is independently C(R³)₂—,or —N(R³)—. In another embodiment, each Z¹ and Z² is independentlyC(R³)₂—, or —N(R³)—; and each R³ is independently H, Me, Et, n-Pr, i-Pr,n-Bu, i-Bu, sec-Bu, n-pentyl, or isopentyl. In a particular embodiment,R³ is H.

In one embodiment, with respect to the bolaamphiphilic compound offormula II, III, IV, V, or VI, each Z¹ and Z² is —O—.

In one embodiment, with respect to the bolaamphiphilic compound offormula I, II, III, or IV, each HG¹ and HG² is independently selectedfrom:

wherein:

-   -   X is —NR^(5a)R^(5b), or —N⁺R^(5a)R^(5b)R^(5c); each R^(5a), and        R^(5b) is independently H or substituted or unsubstituted C₁-C₂₀        alkyl or R^(5a) and R^(5b) may join together to form an N        containing substituted or unsubstituted heteroaryl, or        substituted or unsubstituted heterocyclyl;    -   each R^(5c) is independently substituted or unsubstituted C₁-C₂₀        alkyl; each R⁸ is independently H, substituted or unsubstituted        C₁-C₂₀ alkyl, alkoxy, or carboxy;    -   m1 is 0 or 1; and    -   each n13, n14, and n15 is independently an integer from 1-20.

In one embodiment, with respect to the bolaamphiphilic compound offormula I, II, III, or IV, HG¹ and HG² are as defined above, and each m1is 0.

In one embodiment, with respect to the bolaamphiphilic compound offormula I, II, III, or IV, HG¹ and HG² are as defined above, and each m1is 1.

In one embodiment, with respect to the bolaamphiphilic compound offormula I, II, III, or IV, HG¹ and HG² are as defined above, and eachn13 is 1 or 2.

In one embodiment, with respect to the bolaamphiphilic compound offormula I, II, III, or IV, HG¹ and HG² are as defined above, and eachn14 and n15 is independently 1, 2, 3, 4, or 5. In another embodiment,each n14 and n15 is independently 2 or 3.

In one particular embodiment, the bolaamphiphilic compound is a compoundaccording to formula VIIa, VIIb, VIIc, or VIId:

or a pharmaceutically acceptable salt, solvate, hydrate, prodrug,stereoisomer, tautomer, isotopic variant, or N-oxide thereof, or acombination thereof;wherein:

-   -   each X is —NR^(5a)R^(5b), or —N⁺R^(5a)R^(5b)R^(5c); each R^(5a),        and R^(5b) is independently H or substituted or unsubstituted        C₁-C₂₀ alkyl or R^(5a) and R^(5b) may join together to form an N        containing substituted or unsubstituted heteroaryl, or        substituted or unsubstituted heterocyclyl;        -   each R^(5c) is independently substituted or unsubstituted            C₁-C₂₀ alkyl;        -   n10 is an integer from 2-20; and        -   each dotted bond is independently a single or a double bond.        -   In another particular embodiment, the bolaamphiphilic            compound is a compound according to formula VIIIa, VIIIb,            VIIIc, or VIIId:

or a pharmaceutically acceptable salt, solvate, hydrate, prodrug,stereoisomer, tautomer, isotopic variant, or N-oxide thereof, or acombination thereof;wherein:

-   -   each X is —NR^(5a)R^(5b), or —N⁺R^(5a)R^(5b)R^(5c); each R^(5a),        and R^(5b) is independently H or substituted or unsubstituted        C₁-C₂₀ alkyl or R^(5a) and R^(5b) may join together to form an N        containing substituted or unsubstituted heteroaryl, or        substituted or unsubstituted heterocyclyl;        -   each R^(5c) is independently substituted or unsubstituted            C₁-C₂₀ alkyl;        -   n10 is an integer from 2-20; and        -   each dotted bond is independently a single or a double bond.

In another particular embodiment, the bolaamphiphilic compound is acompound according to formula IXa, IXb, or IXc:

or a pharmaceutically acceptable salt, solvate, hydrate, prodrug,stereoisomer, tautomer, isotopic variant, or N-oxide thereof, or acombination thereof;wherein:

-   -   each X is —NR^(5a)R^(5b), or —N⁺R^(5a)R^(5b)R^(5c); each R^(5a),        and R^(5b) is independently H or substituted or unsubstituted        C₁-C₂₀ alkyl or R^(5a) and R^(5b) may join together to form an N        containing substituted or unsubstituted heteroaryl, or        substituted or unsubstituted heterocyclyl;        -   each R^(5c) is independently substituted or unsubstituted            C₁-C₂₀ alkyl;        -   n10 is an integer from 2-20; and        -   each dotted bond is independently a single or a double bond.

In another particular embodiment, the bolaamphiphilic compound is acompound according to formula Xa, Xb, or Xc:

or a pharmaceutically acceptable salt, solvate, hydrate, prodrug,stereoisomer, tautomer, isotopic variant, or N-oxide thereof, or acombination thereof;wherein:

-   -   each X is —NR^(5a)R^(5b), or —N⁺R^(5a)R^(5b)R^(5c); each R^(5a),        and R^(5b) is independently H or substituted or unsubstituted        C₁-C₂₀ alkyl or R^(5a) and R^(5b) may join together to form an N        containing substituted or unsubstituted heteroaryl, or        substituted or unsubstituted heterocyclyl;        -   each R^(5c) is independently substituted or unsubstituted            C₁-C₂₀ alkyl;        -   n10 is an integer from 2-20; and        -   each dotted bond is independently a single or a double bond.

In one embodiment, with respect to the bolaamphiphilic compound offormula VIIa-VIId, VIIIa-VIIId, IXa-IXc, or Xa-Xc, each dotted bond is asingle bond. In another embodiment, each dotted bond is a double bond.

In one embodiment, with respect to the bolaamphiphilic compound offormula VIIa-VIId, VIIIa-VIIId, IXa-IXc, or Xa-Xc, n10 is an integerfrom 2-16.

In one embodiment, with respect to the bolaamphiphilic compound offormula VIIa-VIId, VIIIa-VIIId, IXa-IXc, or Xa-Xc, n10 is an integerfrom 2-12.

In one embodiment, with respect to the bolaamphiphilic compound offormula VIIa-VIId, VIIIa-VIIId, IXa-IXc, or Xa-Xc, n10 is 2, 4, 6, 8,10, 12, or 16.

In one embodiment, with respect to the bolaamphiphilic compound offormula VIIa-VIId, VIIIa-VIIId, IXa-IXc, or Xa-Xc, each R^(5a), R^(5b),and R^(5c) is independently substituted or unsubstituted C₁-C₂₀ alkyl.

In one embodiment, with respect to the bolaamphiphilic compound offormula VIIa-VIId, VIIIa-VIIId, IXa-IXc, or Xa-Xc, each R^(5a), R^(5b),and R^(5c) is independently unsubstituted C₁-C₂₀ alkyl.

In one embodiment, with respect to the bolaamphiphilic compound offormula VIIa-VIId, VIIIa-VIIId, IXa-IXc, or Xa-Xc, one of R^(5a),R^(5b), and R^(5c) is C₁-C₂₀ alkyl substituted with —OC(O)R⁶; and R⁶ isC₁-C₂₀ alkyl.

In one embodiment, with respect to the bolaamphiphilic compound offormula VIIa-VIId, VIIIa-VIIId, IXa-IXc, or Xa-Xc, two of R^(5a),R^(5b), and R^(5c) are independently C₁-C₂₀ alkyl substituted with—OC(O)R⁶; and R⁶ is C₁-C₂₀ alkyl. In one embodiment, R⁶ is Me, Et, n-Pr,i-Pr, n-Bu, i-Bu, sec-Bu, n-pentyl, isopentyl, n-hexyl, n-heptyl, orn-octyl. In a particular embodiment, R⁶ is Me.

In one embodiment, with respect to the bolaamphiphilic compound offormula VIIa-VIId, VIIIa-VIIId, IXa-IXc, or Xa-Xc, one of R^(5a),R^(5b), and R^(5c) is C₁-C₂₀ alkyl substituted with amino, alkylamino ordialkylamino

In one embodiment, with respect to the bolaamphiphilic compound offormula VIIa-VIId, VIIIa-VIIId, IXa-IXc, or Xa-Xc, two of R^(5a),R^(5b), and R^(5c) are independently C₁-C₂₀ alkyl substituted withamino, alkylamino or dialkylamino

In one embodiment, with respect to the bolaamphiphilic compound offormula VIIa-VIId, VIIIa-VIIId, IXa-IXc, or Xa-Xc, R^(5a), and R^(5b)together with the N they are attached to form substituted orunsubstituted heteroaryl.

In one embodiment, with respect to the bolaamphiphilic compound offormula VIIa-VIId, VIIIa-VIIId, IXa-IXc, or Xa-Xc, R^(5a), and R^(5b)together with the N they are attached to form substituted orunsubstituted pyridyl.

In one embodiment, with respect to the bolaamphiphilic compound offormula VIIa-VIId, VIIIa-VIIId, IXa-IXc, or Xa-Xc, R^(5a), and R^(5b)together with the N they are attached to form substituted orunsubstituted monocyclic or bicyclic heterocyclyl.

In one embodiment, with respect to the bolaamphiphilic compound offormula VIIa-VIId, VIIIa-VIIId, IXa-IXc, or Xa-Xc, X is substituted orunsubstituted

In one embodiment, with respect to the bolaamphiphilic compound offormula VIIa-VIId, VIIIa-VIIId, IXa-IXc, or Xa-Xc, X is

substituted with one or more groups selected from alkoxy, acetyl, andsubstituted or unsubstituted Ph.

In one embodiment, with respect to the bolaamphiphilic compound offormula VIIa-VIId, VIIIa-VIIId, IXa-IXc, or Xa-Xc, X is

In one embodiment, with respect to the bolaamphiphilic compound offormula VIIa-VIId, VIIIa-VIIId, IXa-IXc, or Xa-Xc, X is —NMe₂ or —N⁺Me₃.

In one embodiment, with respect to the bolaamphiphilic compound offormula VIIa-VIId, VIIIa-VIIId, IXa-IXc, or Xa-Xc, X is—N(Me)-CH₂CH₂—OAc or —N⁺(Me)₂—CH₂CF₁₂—OAc.

In one embodiment, with respect to the bolaamphiphilic compound offormula VIIa-VIId, VIIIa-VIIId, IXa-IXc, or Xa-Xc, X is a chitosanylgroup; and the chitosanyl group is a poly-(D)glucosaminyl group with MWof 3800 to 20,000 Daltons, and is attached to the core via N.

In one embodiment, the chitosanyl group is

and wherein each p1 and p2 is independently an integer from 1-400; andeach R^(7a) is H or acyl.

In one embodiment, with respect to the bolaamphiphilic compound offormula I, II, III, IV, V, VI, VIIa-VIIc, VIIIa-VIIIc, IXa-IXc andXa-Xc, the bolaamphiphilic compound is a pharmaceutically acceptablesalt.

In one embodiment, with respect to the bolaamphiphilic compound offormula I, II, III, IV, V, VI, VIIa-VIIc, VIIIa-VIIIc, IXa-IXc andXa-Xc, the bolaamphiphilic compound is in a form of a quaternary salt.

In one embodiment, with respect to the bolaamphiphilic compound offormula I, II, III, IV, V, VI, VIIa-VIIc, VIIIa-VIIIc, IXa-IXc andXa-Xc, the bolaamphiphilic compound is in a form of a quaternary saltwith pharmaceutically acceptable alkyl halide or alkyl tosylate.

In one embodiment, with respect to the bolaamphiphilic compound offormula I, II, III, IV, V, VI, VIIa-VIIc, VIIIa-VIIIc, IXa-IXc andXa-Xc, the bolaamphiphilic compound is any one of the bolaamphiliccompounds listed in Table 1.

In another specific aspect, provided herein are methods forincorporating HIV active drugs in the bolavesicles. In one embodiment,the bolavesicle comprises one or more bolaamphilic compounds describedherein.

The Derivatives and Precursors disclosed can be prepared as illustratedin the Schemes provided herein. The syntheses can involve initialconstruction of, for example, vernonia oil or direct functionalizationof natural derivatives by organic synthesis manipulations such as, butnot limiting to, epoxide ring opening. In those processes involvingoxiranyl ring opening, the epoxy group is opened by the addition ofreagents such as carboxylic acids or organic or inorganic nucleophiles.Such ring opening results in a mixture of two products in which the newgroup is introduced at either of the two carbon atoms of the epoxidemoiety. This provides beta substituted alcohols in which thesubstitution position most remote from the CO group of the mainaliphatic chain of the vernonia oil derivative is arbitrarily assignedas position 1, while the neighboring substituted carbon position isdesignated position 2. For simplicity purposes only, the Derivatives andPrecursors shown herein may indicate structures with the hydroxy groupalways at position 2 but the Derivatives and Precursors wherein thehydroxy is at position 1 are also encompassed by the invention. Thus, aradical of the formula —CH(OH)—CH(R)— refers to the substitution of —OHat either the carbon closer to the CO group, designated position 2 or tothe carbon at position 1. Moreover, with respect to the preparation ofsymmetrical bolaamphiphiles made via introducing the head groups throughan epoxy moiety (e.g., as in vernolic acid) or a double bond (—C═C—) asin mono unsaturated fatty acids (e.g., oleic acid) a mixture of threedifferent derivatives will be produced. In certain embodiments, vesiclesare prepared using the mixture of unfractionated positional isomers. Inone aspect of this embodiment, where one or more bolas are prepared fromvernolic acid, and in which a hydroxy group as well as the head groupintroduced through an epoxy or a fatty acid with the head groupintroduced through a double bond (—C═C—), the bola used in vesiclepreparation can actually be a mixture of three different positionalisomers.

In other embodiments, the three different derivatives are isolated.Accordingly, the vesicles disclosed herein can be made from a mixture ofthe three isomers of each derivative or, in other embodiments, theindividual isomers can be isolated and used for preparation of vesicles.

Symmetrical bolaamphiphiles can form relatively stable self aggregatevesicle structures by the use of additives such as cholesterol andcholesterol derivatives (e.g., cholesterol hemisuccinate, cholesterololeyl ether, anionic and cationic derivatives of cholesterol and thelike), or other additives including single headed amphiphiles with one,two or multiple aliphatic chains such as phospholipids, zwitterionic,acidic, or cationic lipids. Examples of zwitterionic lipids arephosphatidylcholines, phosphatidylethanol amines and sphingomyelins.Examples of acidic amphiphilic lipids are phosphatidylglycerols,phosphatidylserines, phosphatidylinositols, and phosphatidic acids.Examples of cationic amphipathic lipids are diacyl trimethylammoniumpropanes, diacyl dimethylammonium propanes, and stearylamines cationicamphiphiles such as spermine cholesterol carbamates, and the like, inoptimum concentrations which fill in the larger spaces on the outersurfaces, and/or add additional hydrophilicity to the particles. Suchadditives may be added to the reaction mixture during formation ofnanoparticles to enhance stability of the nanoparticles by filling inthe void volumes of in the upper surface of the vesicle membrane.

Stability of nano vesicles according to the present disclosure can bedemonstrated by dynamic light scattering (DLS) and transmission electronmicroscopy (TEM). For example, suspensions of the vesicles can be leftto stand for 1, 5, 10, and 30 days to assess the stability of thenanoparticle solution/suspension and then analyzed by DLS and TEM.

The vesicles disclosed herein may encapsulate within their core theactive agent, which in particular embodiments is selected from peptides,proteins, nucleotides and or non-polymeric agents. In certainembodiments, the active agent is also associated via one or morenon-covalent interactions to the vesicular membrane on the outer surfaceand/or the inner surface, optionally as pendant decorating the outer orinner surface, and may further be incorporated into the membranesurrounding the core. In certain aspects, biologically active peptides,proteins, nucleotides or non-polymeric agents that have a net electriccharge, may associate ionically with oppositely charged headgroups onthe vesicle surface and/or form salt complexes therewith.

In particular aspects of these embodiments, additives which may bebolaamphiphiles or single headed amphiphiles, comprise one or morebranching alkyl chains bearing polar or ionic pendants, wherein thealiphatic portions act as anchors into the vesicle's membrane and thependants (e.g., chitosan derivatives or polyamines or certain peptides)decorate the surface of the vesicle to enhance penetration throughvarious biological barriers such as the intestinal tract and the BBB,and in some instances are also selectively hydrolyzed at a given site orwithin a given organ. The concentration of these additives is readilyadjusted according to experimental determination.

In certain embodiments, the oral formulations of the present disclosurecomprise agents that enhance penetration through the membranes of the GItract and enable passage of intact nanoparticles containing the drug.These agents may be any of the additives mentioned above and, inparticular aspects of these embodiment, include chitosan and derivativesthereof, serving as vehicle surface ligands, as decorations or pendantson the vesicles, or the agents may be excipients added to theformulation.

In other embodiments, the nanoparticles and vesicles disclosed hereinmay comprise the fluorescent marker carboxyfluorescein (CF) encapsulatedtherein while in particular aspects, the nanoparticle and vesicles ofthe present disclosure may be decorated with one or more of PEG, e.g.PEG2000-vernonia derivatives as pendants. For example, two kinds ofPEG-vernonia derivatives can be used: PEG-ether derivatives, wherein PEGis bound via an ether bond to the oxygen of the opened epoxy ring of,e.g., vernolic acid and PEG-ester derivatives, wherein PEG is bound viaan ester bond to the carboxylic group of, e.g., vernolic acid.

In other embodiments, vesicles, made from synthetic amphiphiles, as wellas liposomes, made from synthetic or natural phospholipids,substantially (or totally) isolate the therapeutic agent from theenvironment allowing each vesicle or liposome to deliver many moleculesof the therapeutic agent. Moreover, the surface properties of thevesicle or liposome can be modified for biological stability, enhancedpenetration through biological barriers and targeting, independent ofthe physico-chemical properties of the encapsulated drug.

In still other embodiments, the headgroup is selected from: (i) cholineor thiocholine, O-alkyl, N-alkyl or ester derivatives thereof; (ii)non-aromatic amino acids with functional side chains such as glutamicacid, aspartic acid, lysine or cysteine, or an aromatic amino acid suchas tyrosine, tryptophan, phenylalanine and derivatives thereof such aslevodopa (3,4-dihydroxy-phenylalanine) and p-aminophenylalanine; (iii) apeptide or a peptide derivative that is specifically cleaved by anenzyme at a diseased site selected from enkephalin, N-acetyl-ala-ala, apeptide that constitutes a domain recognized by beta and gammasecretases, and a peptide that is recognized by stromelysins; (iv)saccharides such as glucose, mannose and ascorbic acid; and (v) othercompounds such as nicotine, cytosine, lobeline, polyethylene glycol, acannabinoid, or folic acid.

In further embodiments, nano-sized particle and vesicles disclosedherein further comprise at least one additive for one or more oftargeting purposes, enhancing permeability and increasing the stabilitythe vesicle or particle. Such additives, in particular aspects, mayselected from: (i) a single headed amphiphilic derivative comprisingone, two or multiple aliphatic chains, preferably two aliphatic chainslinked to a midsection/spacer region such as —NH—(CH₂)₂—N—(CH₂)₂—N—, or—O—(CH₂)₂—N—(CH₂)₂—O—, and a sole headgroup, which may be a selectivelycleavable headgroup or one containing a polar or ionic selectivelycleavable group or moiety, attached to the N atom in the middle of saidmidsection. In other aspects, the additive can be selected from amongcholesterol and cholesterol derivatives such as cholesterylhemmisuccinate; phospholipids, zwitterionic, acidic, or cationic lipids;chitosan and chitosan derivatives, such as vernolic acid-chitosanconjugate, quaternized chitosan, chitosan-polyethylene glycol (PEG)conjugates, chitosan-polypropylene glycol (PPG) conjugates, chitosanN-conjugated with different amino acids, carboxyalkylated chitosan,sulfonyl chitosan, carbohydrate-branched N-(carboxymethylidene) chitosanand N-(carboxymethyl) chitosan; polyamines such as protamine, polylysineor polyarginine; ligands of specific receptors at a target site of abiological environment such as nicotine, cytisine, lobeline, 1-glutamicacid MK801, morphine, enkephalins, benzodiazepines such as diazepam(valium) and librium, dopamine agonists, dopamine antagonists tricyclicantidepressants, muscarinic agonists, muscarinic antagonists,cannabinoids and arachidonyl ethanol amide; polycationic polymers suchas polyethylene amine; peptides that enhance transport through the BBBsuch as OX 26, transferrins, polybrene, histone, cationic dendrimer,synthetic peptides and polymyxin B nonapeptide (PMBN); monosaccharidessuch as glucose, mannose, ascorbic acid and derivatives thereof;modified proteins or antibodies that undergo absorptive-mediated orreceptor-mediated transcytosis through the blood-brain barrier, such asbradykinin B2 agonist RMP-7 or monoclonal antibody to the transferrinreceptor; mucoadhesive polymers such as glycerides and steroidaldetergents; and Ca²⁺ chelators. The aforementioned head groups on theadditives designed for one or more of targeting purposes and enhancingpermeability may also be a head group, preferably on an asymmetricbolaamphiphile wherein the other head group is another moiety, or thehead group on both sides of a symmetrical bolaamphiphile.

In other embodiments, nano-sized particle and vesicles discloser hereinmay comprises at least one biologically active agent is selected from:(i) a natural or synthetic peptide or protein such as analgesicspeptides from the enkephalin class, insulin, insulin analogs, oxytocin,calcitonin, tyrotropin releasing hormone, follicle stimulating hormone,luteinizing hormone, vasopressin and vasopressin analogs, catalase,interleukin-II, interferon, colony stimulating factor, tumor necrosisfactor (TNF), melanocyte-stimulating hormone, superoxide dismutase,glial cell derived neurotrophic factor (GDNF) or the Gly-Leu-Phe (GLF)families; (ii) nucleosides and polynucleotides selected from DNA or RNAmolecules such as small interfering RNA (siRNA) or a DNA plasmid; (iii)antiviral and antibacterial; (iv) antineoplastic and chemotherapy agentssuch as cyclosporin, doxorubicin, epirubicin, bleomycin, cisplatin,carboplatin, vinca alkaloids, e.g. vincristine, Podophyllotoxin,taxanes, e.g. Taxol and Docetaxel, and topoisomerase inhibitors, e.g.irinotecan, topotecan.

In another specific aspect, provided herein are methods forbrain-targeted drug delivery using the bolavesicles incorporated withHIV active drug.

In one particular embodiment, the HIV active drug is Tenofovir or({[(2R)-1-(6-amino-9H-purin-9-yl)propan-2-yl]oxy}methyl)phosphonic acid.

In another particular embodiment, the HIV active drug is fosamprenavir.In another particular embodiment, the HIV active drug is enfuvirtide. Inanother particular embodiment, the HIV active drug is saquinavir. Inanother particular embodiment, the HIV active drug is lamivudine. Inanother particular embodiment, the HIV active drug is stavudine.

In another specific aspect, provided herein are methods for deliveringTenofovir to the brain.

In another specific aspect, provided herein are methods for deliveringfosamprenavir, enfuvirtide, saquinavir, lamivudine or stavudine to thebrain.

In another specific aspect, provided herein are nano-particles,comprising one or more bolaamphiphilic compounds and Tenofovir,fosamprenavir, enfuvirtide, saquinavir, lamivudine or stavudine. In oneembodiment, the bolaamphiphilic compounds and Tenofovir, fosamprenavir,enfuvirtide, saquinavir, lamivudine or stavudine are encapsulated withinthe nano-particle.

In another specific aspect, provided herein are pharmaceuticalcompositions, comprising a nano-sized particle comprising one or morebolaamphiphilic compounds and Tenofovir, fosamprenavir, enfuvirtide,saquinavir, lamivudine or stavudine; and a pharmaceutically acceptablecarrier.

In another specific aspect, provided herein are methods for treatment ordiagnosis of diseases or disorders selected from HIV and relateddiseases using the nano-particles, pharmaceutical compositions orformulations of the present invention.

In certain embodiments, vesicle formation is carried out in the presenceof an additive that increases encapsulation of a therapeutic agent. Inone aspect of this embodiment, the additive is stearyl amine. In anotheraspect, the therapeutic agent is an antiviral agent, while in a stillfurther aspect the antiviral agent is Tenofovir.

In certain embodiments, the present disclosure is directed to a methodof treatment of a patient afflicted with a viral disease comprisingadministration of vesicles described herein. In one aspect of thisembodiment, the patient is a human AIDS patient in need of suchtreatment. In particular aspects of this embodiment, the vesicles areformed in the presence of an additive that increases encapsulation of atherapeutic agent. In one aspect of this embodiment, the additive isstearyl amine. In another aspect, the therapeutic agent is an antiviralagent, while in a still further aspect the antiviral agent is Tenofovir.In another aspect of this embodiment, the virus is HIV.

Additional embodiments within the scope provided herein are set forth innon-limiting fashion elsewhere herein and in the examples. It should beunderstood that these examples are for illustrative purposes only andare not to be construed as limiting in any manner

Pharmaceutical Compositions

In another aspect, the invention provides a pharmaceutical compositioncomprising a pharmaceutically acceptable carrier and a pharmaceuticallyeffective amount of a compound of Formula I or a complex thereof.

When employed as pharmaceuticals, the compounds provided herein aretypically administered in the form of a pharmaceutical composition. Suchcompositions can be prepared in a manner well known in thepharmaceutical art and comprise at least one active compound.

In certain embodiments, with respect to the pharmaceutical composition,the carrier is a parenteral carrier, oral or topical carrier.

The present invention also relates to a compound or pharmaceuticalcomposition of compound according to Formula I; or a pharmaceuticallyacceptable salt or solvate thereof for use as a pharmaceutical or amedicament.

Generally, the compounds provided herein are administered in atherapeutically effective amount. The amount of the compound actuallyadministered will typically be determined by a physician, in the lightof the relevant circumstances, including the condition to be treated,the chosen route of administration, the actual compound administered,the age, weight, and response of the individual patient, the severity ofthe patient's symptoms, and the like.

The pharmaceutical compositions provided herein can be administered by avariety of routes including oral, rectal, transdermal, subcutaneous,intravenous, intramuscular, and intranasal. Depending on the intendedroute of delivery, the compounds provided herein are preferablyformulated as either injectable or oral compositions or as salves, aslotions or as patches all for transdermal administration.

The compositions for oral administration can take the form of bulkliquid solutions or suspensions, or bulk powders. More commonly,however, the compositions are presented in unit dosage forms tofacilitate accurate dosing. The term “unit dosage forms” refers tophysically discrete units suitable as unitary dosages for human subjectsand other mammals, each unit containing a predetermined quantity ofactive material calculated to produce the desired therapeutic effect, inassociation with a suitable pharmaceutical excipient. Typical unitdosage forms include prefilled, premeasured ampules or syringes of theliquid compositions or pills, tablets, capsules or the like in the caseof solid compositions. In such compositions, the compound is usually aminor component (from about 0.1 to about 50% by weight or preferablyfrom about 1 to about 40% by weight) with the remainder being variousvehicles or carriers and processing aids helpful for forming the desireddosing form.

Liquid forms suitable for oral administration may include a suitableaqueous or nonaqueous vehicle with buffers, suspending and dispensingagents, colorants, flavors and the like. Solid forms may include, forexample, any of the following ingredients, or compounds of a similarnature: a binder such as microcrystalline cellulose, gum tragacanth orgelatin; an excipient such as starch or lactose, a disintegrating agentsuch as alginic acid, Primogel, or corn starch; a lubricant such asmagnesium stearate; a glidant such as colloidal silicon dioxide; asweetening agent such as sucrose or saccharin; or a flavoring agent suchas peppermint, methyl salicylate, or orange flavoring.

Injectable compositions are typically based upon injectable sterilesaline or phosphate-buffered saline or other injectable carriers knownin the art. As before, the active compound in such compositions istypically a minor component, often being from about 0.05 to 10% byweight with the remainder being the injectable carrier and the like.

Transdermal compositions are typically formulated as a topical ointmentor cream containing the active ingredient(s), generally in an amountranging from about 0.01 to about 20% by weight, preferably from about0.1 to about 20% by weight, preferably from about 0.1 to about 10% byweight, and more preferably from about 0.5 to about 15% by weight. Whenformulated as a ointment, the active ingredients will typically becombined with either a paraffinic or a water-miscible ointment base.Alternatively, the active ingredients may be formulated in a cream with,for example an oil-in-water cream base. Such transdermal formulationsare well-known in the art and generally include additional ingredientsto enhance the dermal penetration of stability of the active ingredientsor the formulation. All such known transdermal formulations andingredients are included within the scope provided herein.

The compounds provided herein can also be administered by a transdermaldevice. Accordingly, transdermal administration can be accomplishedusing a patch either of the reservoir or porous membrane type, or of asolid matrix variety.

The above-described components for orally administrable, injectable ortopically administrable compositions are merely representative. Othermaterials as well as processing techniques and the like are set forth inPart 8 of Remington's Pharmaceutical Sciences, 17th edition, 1985, MackPublishing Company, Easton, Pa., which is incorporated herein byreference.

The above-described components for orally administrable, injectable, ortopically administrable compositions are merely representative. Othermaterials as well as processing techniques and the like are set forth inPart 8 of Remington's The Science and Practice of Pharmacy, 21stedition, 2005, Publisher: Lippincott Williams & Wilkins, which isincorporated herein by reference.

The compounds of this invention can also be administered in sustainedrelease forms or from sustained release drug delivery systems. Adescription of representative sustained release materials can be foundin Remington's Pharmaceutical Sciences.

The present invention also relates to the pharmaceutically acceptableformulations of compounds of Formula I. In certain embodiments, theformulation comprises water. In another embodiment, the formulationcomprises a cyclodextrin derivative. In certain embodiments, theformulation comprises hexapropyl-β-cyclodextrin. In a more particularembodiment, the formulation comprises hexapropyl-β-cyclodextrin (10-50%in water).

The present invention also relates to the pharmaceutically acceptableacid addition salts of compounds of Formula I. The acids which are usedto prepare the pharmaceutically acceptable salts are those which formnon-toxic acid addition salts, i.e. salts containing pharmacologicallyacceptable anioνs such as the hydrochloride, hydroiodide, hydrobromide,nitrate, sulfate, bisulfate, πhosphate, acetate, lactate, citrate,tartrate, succinate, maleate, fumarate, benzoate, para-toluenesulfonate,and the like.

The following formulation examples illustrate representativepharmaceutical compositions that may be prepared in accordance with thisinvention. The present invention, however, is not limited to thefollowing pharmaceutical compositions.

Formulation 1 Injection

A compound of the invention may be dissolved or suspended in a bufferedsterile saline injectable aqueous medium to a concentration ofapproximately 5 mg/mL.

In particular embodiments, the formulations of the disclosure comprisevesicles prepared as described herein. In certain embodiments, suchvesicle formation comprise an additive that increases encapsulation of atherapeutic agent. In one aspect of this embodiment, the additive isstearyl amine. In another aspect, the therapeutic agent is an antiviralagent, while in a still further aspect the antiviral agent is Tenofovir.

Methods of Treatment

Bolaamphiphilic vesicles (bolavesicles) may have certain advantages overconventional liposomes as potential vehicles for drug delivery.Bolavesicles have thinner membranes than comparable liposomal bilayer,and therefore possess bigger inner volume and hence higher encapsulationcapacity than liposomes of the same diameter. Moreover, bolavesicles aremore physically-stable than conventional liposomes in part because ofreduced limit exchange, but can be destabilized in a triggered fashion(e.g., by hydrolysis of the headgroups using a specific enzymaticreaction) thus allowing controlled release of the encapsulated materialat the site of action (i.e., drug targeting). In still another aspectthe vesicles formed from the bolaamphiphiles contain additives that helpto stabilize the vesicles, by stabilizing the vesicle's membranes, suchas but not limited to cholesterol derivatives such as cholesterylhemisuccinate and cholesterol itself and combinations such ascholesteryl hemisuccinate and cholesterol. In still another embodimentsthe vesicles in addition to these components have another additiveswhich decorates the outer vesicle membranes with groups or pendants thatenhance penetration though biological barriers such as the BBB, orgroups for targeting to specific sites.

Thus, various HIV active drug molecules can be encapsulated in thebolaamphiphilic vesicles and then delivered to the brain in sufficientconcentrations for therapeutic use.

The bolaamphiphiles aggregate into encapsulating monolayer membraneswhich, together with additional additives for stability and additivesfor functional surface groups, provide vesicle stability, penetrabilitythrough the BBB and a controlled release mechanism that enables therelease of the encapsulated drug primarily in the brain.

General Synthetic Procedures

The compounds provided herein can be purchased or prepared from readilyavailable starting materials using the following general methods andprocedures. See, e.g., Synthetic Schemes below. It will be appreciatedthat where typical or preferred process conditions (i.e., reactiontemperatures, times, mole ratios of reactants, solvents, pressures,etc.) are given, other process conditions can also be used unlessotherwise stated. Optimum reaction conditions may vary with theparticular reactants or solvent used, but such conditions can bedetermined by one skilled in the art by routine optimization procedures.

Additionally, as will be apparent to those skilled in the art,conventional protecting groups may be necessary to prevent certainfunctional groups from undergoing undesired reactions. The choice of asuitable protecting group for a particular functional group as well assuitable conditions for protection and deprotection are well known inthe art. For example, numerous protecting groups, and their introductionand removal, are described in T. W. Greene and P. G. M. Wuts, ProtectingGroups in Organic Synthesis, Second Edition, Wiley, New York, 1991, andreferences cited therein.

The compounds provided herein may be isolated and purified by knownstandard procedures. Such procedures include (but are not limited to)recrystallization, column chromatography or HPLC. The following schemesare presented with details as to the preparation of representativesubstituted biarylamides that have been listed herein. The compoundsprovided herein may be prepared from known or commercially availablestarting materials and reagents by one skilled in the art of organicsynthesis.

The enantiomerically pure compounds may be prepared according to anytechniques known to those of skill in the art. For instance, they may beprepared by chiral or asymmetric synthesis from a suitable opticallypure precursor or obtained from a racemate by any conventionaltechnique, for example, by chromatographic resolution using a chiralcolumn, TLC or by the preparation of diastereoisomers, separationthereof and regeneration of the desired enantiomer. See, e.g.,“Enantiomers, Racemates and Resolutions,” by J. Jacques, A. Collet, andS. H. Wilen, (Wiley-Interscience, New York, 1981); S. H. Wilen, A.Collet, and J. Jacques, Tetrahedron, 2725 (1977); E. L. ElielStereochemistry of Carbon Compounds (McGraw-Hill, NY, 1962); and S. H.Wilen Tables of Resolving Agents and Optical Resolutions 268 (E. L.Eliel ed., Univ. of Notre Dame Press, Notre Dame, 1N, 1972,Stereochemistry of Organic Compounds, Ernest L. Eliel, Samuel H. Wilenand Lewis N. Manda (1994 John Wiley & Sons, Inc.), and StereoselectiveSynthesis A Practical Approach, Mihály Nógrádi (1995 VCH Publishers,Inc., NY, N.Y.).

In certain embodiments, an enantiomerically pure compound of formula (I)may be obtained by reaction of the racemate with a suitable opticallyactive acid or base. Suitable acids or bases include those described inBighley et al., 1995, Salt Forms of Drugs and Adsorption, inEncyclopedia of Pharmaceutical Technology, vol. 13, Swarbrick & Boylan,eds., Marcel Dekker, New York; ten Hoeve & H. Wynberg, 1985, Journal ofOrganic Chemistry 50:4508-4514; Dale & Mosher, 1973, J. Am. Chem. Soc.95:512; and CRC Handbook of Optical Resolution via Diastereomeric SaltFormation, the contents of which are hereby incorporated by reference intheir entireties.

Enantiomerically pure compounds can also be recovered either from thecrystallized diastereomer or from the mother liquor, depending on thesolubility properties of the particular acid resolving agent employedand the particular acid enantiomer used. The identity and optical purityof the particular compound so recovered can be determined by polarimetryor other analytical methods known in the art. The diastereoisomers canthen be separated, for example, by chromatography or fractionalcrystallization, and the desired enantiomer regenerated by treatmentwith an appropriate base or acid. The other enantiomer may be obtainedfrom the racemate in a similar manner or worked up from the liquors ofthe first separation.

In certain embodiments, enantiomerically pure compound can be separatedfrom racemic compound by chiral chromatography. Various chiral columnsand eluents for use in the separation of the enantiomers are availableand suitable conditions for the separation can be empirically determinedby methods known to one of skill in the art. Exemplary chiral columnsavailable for use in the separation of the enantiomers provided hereininclude, but are not limited to CHIRALCEL® OB, CHIRALCEL® OB-H,CHIRALCEL® OD, CHIRALCEL® OD-H, CHIRALCEL® OF, CHIRALCEL® OG, CHIRALCEL®OJ and CHIRALCEL® OK.

ABBREVIATIONS

-   -   BBB, blood brain barrier    -   BCECs, brain capillary endothelial cells    -   CF, carboxyfluorescein    -   CHEMS, cholesteryl hemisuccinate    -   CHOL, cholesterol    -   Cryo-TEM, Cryo-transmission electron microscope    -   DAPI, 4′,6-diamidino-2-phenylindole    -   DDS, drug delivery system    -   DLS, dynamic light scattering    -   DMPC, 1,2-dimyristoyl-sn-glycero-3-phosphocholine    -   DMPE, 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine    -   DMPG,1,2-dimyristoyl-sn-glycero-3-phospho-(1′-rac-glycerol)    -   EPR, electron paramagnetic resonance    -   FACS, fluorescence-activated cell sorting    -   FCR, fluorescence colorimetric response    -   GUVs, giant unilamellar vesicles    -   HPLC, high performance liquid chromatography    -   IR, infrared    -   MRI, magnetic resonance imaging    -   NMR, nuclear magnetic resonance    -   NPs, nanoparticles    -   PBS, phosphate buffered saline    -   PC, phosphatidylcholine    -   PDA, polydiacetylene.    -   TMA-DPH, 1-(4 trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene

Example 1 Bolaamphiphile Synthesis

The boloamphiphles or bolaamphiphilic compounds of the invention can besynthesized following the procedures described previously (see below).

Briefly, the carboxylic group of methyl vernolate or vernolic acid wasinteracted with aliphatic diols to obtain bisvernolesters. Then theepoxy group of the vernolate moiety, located on C12 and C13 of thealiphatic chain of vernolic acid, was used to introduce two AChheadgroups on the two vicinal carbons obtained after the opening of theoxirane ring. For GLH-20 (Table 1), the ACh head group was attached tothe vernolate skeleton through the nitrogen atom of the choline moiety.The bolaamphiphile was prepared in a two-stage synthesis: First, openingof the epoxy ring with a haloacetic acid and, second, quaternizationwith the N,N-dimethylamino ethyl acetate. For GLH-19 (Table 1) thatcontains an ACh head group attached to the vernolate skeleton throughthe acetyl group, the bolaamphiphile was prepared in a three-stagesynthesis, including opening of the epoxy ring with glutaric acid, thenesterification of the free carboxylic group with N,N-dimethyl aminoethanol and the final product was obtained by quaternization of the headgroup, using methyl iodide followed by exchange of the iodide ion bychloride using an ion exchange resin.

Each bolaamphiphile was characterized by mass spectrometry, NMR and IRspectroscopy. The purity of the two bolaamphiphiles was >97% asdetermined by HPLC.

Materials. Iron(III) acetylacetonate (Fe(acac)₃), diphenyl ether,1,2-hexadecanediol, oleic acid, oleylamine, and carboxyfluorescein (CF)were purchased from Sigma Aldrich (Rehovot, Israel). Chloroform andethanol were purchased from Bio-Lab Ltd. Jerusalem, Israel.1,2-dimyristoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (DMPG),1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine(DMPE),1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), cholesterol(CHOL), cholesteryl hemisuccinate (CHEMS) were purchased from AvantiLipids (Alabaster, Ala., USA), The diacetylenic monomer10,12-tricosadiynoic acid was purchased from Alfa Aesar (Karlsruhe,Germany), and purified by dissolving the powder in chloroform, filteringthe resulting solution through a 0.45 μm nylon filter (Whatman Inc.,Clifton, N.J., USA), and evaporation of the solvent. 1-(4trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH) waspurchased from Molecular Probes Inc. (Eugene, Oreg., USA).

Synthesis of Representative Bolaamphiphilic Compounds

The synthesis bolaamphiphilic compounds of this invention can be carriedout in accordance with the methods described previously (Chemistry andPhysics of Lipids 2008, 153, 85-97; Journal of Liposome Research 2010,20, 147-59; WO2002/055011; WO2003/047499; or WO2010/128504) and usingthe appropriate reagents, starting materials, and purification methodsknown to those skilled in the art. Table 1 lists the representativebolaamphiphilic compounds of the invention.

TABLE 1 Representative Bolaamphiphiles # Structure GLH-3

GLH-4

GLH-5

GLH-6 ^(a)

GLH-7

GLH-8

GLH-9

GLH-10

GLH-11

GLH-12 ^(a)

GLH-13 ^(a)

GLH-13 ^(a)

GLH-14

GLH-15

GLH-16

GLH-17

GLH-18

GLH-19

GLH-20

GLH-21

GLH-22

GLH-23

GLH-24

GLH-25

GLH-26

GLH-27

GLH-28

GLH-29

GLH-30

GLH-30

GLH-31

GLH-32

GLH-33

GLH-34

GLH-35

GLH-36

GLH-37

GLH-38

GLH-39^(a)

GLH-40

GLH-41

GLH-42 ^(a)

GLH-43 ^(a)

GLH-44

GLH-45

GLH-46

GLH-47

GLH-48

GLH-49 ^(a)

GLH-50^(a)

GLH-51 ^(a)

GLH-52^(a)

GLH-53 ^(a)

GLH-54 ^(a)

GLH-55

GLH-56

GLH-57

^(a) -an intermediate

Example 2 Bolavesicle Preparation and Characterization

Bolaamphiphiles, cholesterol, and CHMES (2:1:1 mole ratio) are dissolvedin chloroform or a suitable solvent. 0.5 mg of the HIV active drugdispersed in chloroform is added to the mix. The solvents are evaporatedunder vacuum and the resultant thin films are hydrated in 0.2 mg/mL CFsolution in PBS and probe-sonicated (Vibra-Cell VCX130 sonicator, Sonicsand Materials Inc., Newtown, Conn., USA) with amplitude 20%, pulse on:15 sec, pulse off: 10 sec to achieve homogenous vesicle dispersions.Vesicle size and zeta potential were determined using a Zetasizer NanoZS (Malvern Instruments, UK). The amount of the HIV active drugencapsulated in the vesicles can be determined by HPLC and/or UVspectroscopy (G Gnanarajan, et al, 2009) after separating thenon-encapsulated drug, by size exclusion chromatography (onSephadex-G50).

Spectral Characterization Example 3 Electron Paramagnetic Resonance(EPR)

EPR spectra of HIV active drug embedded bolavesicles resuspended in PBScan be obtained using a Bruker EMX-220 X-band (υ-9.4 GHz) EPRspectrometer equipped with an Oxford Instruments ESR 900 temperatureaccessories and an Agilent 53150A frequency counter. Spectra can berecorded at room temperature with the non-saturating incident microwavepower 20 mW and the 100 KHz magnetic field modulation of 0.2 mTamplitude. Processing of EPR spectra, determination of spectralparameters can be done using Bruker WIN-EPR software.

Example 4 Cryogenic Transmission Electron Microscopy (Cryo-TEM)

Specimens studied by cryo-TEM were prepared. Sample solutions (4 μL) aredeposited on a glow discharged, 300 mesh, lacey carbon copper grids (TedPella, Redding, Calif., USA). The excess liquid is blotted and thespecimen was vitrified in a Leica EM GP vitrification system in whichthe temperature and relative humidity are controlled. The samples areexamined at −180° C. using a FEI Tecnai 12 G2 TWIN TEM equipped with aGatan 626 cold stage, and the images are recorded (Gatan model 794charge-coupled device camera) at 120 kV in low-dose mode. FIG. 1 showsTEM micrograph of vesicles from GLH-20 (A) and their size distributiondetermined by DLS (B).

Assays Example 5 Lipid/polydiacetylene (PDA) Assay

Lipid/polydiacetylene (PDA) vesicles (PDADMPC 3:2, mole ratio) areprepared by dissolving the lipid components in chloroform/ethanol anddrying together in vacuo. Vesicles are subsequently prepared in DDW byprobe-sonication of the aqueous mixture at 70° C. for 3 min. The vesiclesamples are then cooled at room temperature for an hour and kept at 4°C. overnight. The vesicles are then polymerized using irradiation at 254nm for 10-20 s, with the resulting emulsions exhibiting an intense blueappearance. PDA fluorescence is measured in 96-well microplates (GreinerBio-One GmbH, Frickenhausen, Germany) on a Fluoroscan Ascentfluorescence plate reader (Thermo Vantaa, Finland). All measurements areperformed at room temperature at 485 nm excitation and 555 nm emissionusing LP filters with normal slits. Acquisition of data is automaticallyperformed every 5 min for 60 min. Samples comprised 30 μL of DMPCPDAvesicles and 5 μL bolaamphiphilic vesicles assembled with HIV drug,followed by addition of 30 μL 50 mM Tris-base buffer (pH 8.0).

A quantitative value for the increasing of the fluorescence intensitywithin the PDA/PC-labeled vesicles is given by the fluorescencecolorimetric response (% FCR), which is defined as follows²⁷:

%FCR=[(F _(I) −F ₀)/F ₁₀₀]·100  Eq. 1.

Where F_(I) is the fluorescence emission of the lipid/PDA vesicles afteraddition of the tested membrane-active compounds, F₀ is the fluorescenceof the control sample (without addition of the compounds), and F₁₀₀ isthe fluorescence of a sample heated to produce the highest fluorescenceemission of the red PDA phase minus the fluorescence of the controlsample.

Example 6 Cell Culture

b.End3 immortalized mouse brain capillary endothelium cells are kindlyprovided by Prof. Philip Lazarovici (Institute for Drug Research, Schoolof Pharmacy, The Hebrew University of Jerusalem, Israel). The b.End3cells were cultured in DMEM medium supplemented with 10% fetal bovineserum, 2 mM L-Glutamine, 100 IU/mL penicillin and 100 mg/mL streptomycin(Biological Industries Ltd., Beit Haemek, Israel). The cells aremaintained in an incubator at 37° C. in a humidified atmosphere with 5%CO₂.

Example 7 Internalization of CF by the Cells In Vitro

b.End3 cells are grown on 24-well plates or on coverslips (for FACS andfluorescence microscopy analysis, respectively). The medium is replacedwith culture medium without serum and CF solution, or testedbolavesicles (equivalent to 0.5 mg/mL CF), or equivalent volume of themedium are added to the cells and incubated for 5 hr at 4° C. or at 37°C. At the end of the incubation, cells are extensively washed withcomplete medium and with PBS, and are either detached from the platesusing trypsin-EDTA solution (Biological Industries Ltd., Beit Haemek,Israel) and analyzed by FACS (FACSCalibur Flow Cytometer, BDBiosciences, USA), or fixed with 2.5% formaldehyde in PBS, washed twicewith PBS, mounted on slides using Mowiol-based mounting solution andanalyzed using a FV1000-IX81 confocal microscope (Olympus, Tokyo, Japan)equipped with 60× objective. All the images are acquired using the sameimaging settings and are not corrected or modified. The FIG. 2 showshead group hydrolysis by AChE (A) of GLH-19 (blue) and GLH-20 (red) andrelease of CF from GLH-19 vesicles (B) and GLH-20 vesicles (C). AChEcauses the release of encapsulated material from GLH-20 vesicles, butnot from GLH-19 vesicles (FIG. 2). The vesicles are capable ofdelivering small molecules, such as carboxyfluorescein (CF), into amouse brain, but the fluorescent dye accumulates only if it is deliveredin vesicles that release their encapsulated CF in presence of AChE,namely, GLH-20 vesicles (FIG. 3A). These results suggest that therelease is due to head group hydrolysis by AChE in the brain.Corroboration for this conclusion also comes from an experiment showingthat when an analgesic peptide is delivered to the brain by the bolavesicles, analgesia (which is caused when the encapsulated peptide isreleased in the brain) was observed only with GLH-20 vesicles, but notby GLH-19 vesicles (FIG. 4A).The vesicles do not break the BBB, butrather penetrate it in their intact form, as indicated by the findingthat analgesia is obtained only when enkephalin is administered whileencapsulated within the vesicles, but not when free enkephalin isadministered together with empty vesicles (FIG. 4B).

The ACh head groups also provide the vesicles with cationic surfaces,which promote penetration through the BBB [Lu et al, 2005] and transportof the encapsulated material into the brain. Toxicity studies showedthat the dose which induced the first toxic signs was 10-20 times higherthan the doses needed to obtain analgesia by encapsulated analgesicpeptides.

The addition of chitosan (CS) surface groups, by employing CS-vernolateconjugates, increased BBB permeability of the vesicles (FIG. 4B),probably by increasing transcytosis [Newton, 2006]. However, the CSgroups, when added to the vesicles by employing fatty acid-CS conjugate(in this case, vernolic acid), are not stable in circulation as surfacegroups because of the low energy barrier for lipid exchange of suchconjugates.

In addition to the peptide leu-enkephalin, and the small molecules: CF,uranyl acetate, kyotorphin and sucrose, can also be successfullyencapsulated in these vesicles the proteins albumin and trypsinogen andthe polysaccharide Dextran-FITC (MW 9000). Albumin-FITC, encapsulated,was delivered successfully to the brain (FIG. 5B), while un-encapsulatedalbumin-FITC showed little, if any, brain accumulation (FIG. 5A),indicating that the vesicle transported the protein into the brainthrough the BBB. These results strongly suggest that the vesicles can bemade to encapsulate other molecules, such as anti-retroviral drugs, anddeliver them into the brain without harming the BBB.

Example 8 Statistical Analysis

The data are presented as mean and standard deviations (SD) or standarderrors of mean (SEM). Statistical differences between the control andthe studied formulations are analyzed using ANOVA followed by Dunnettpost-test using InStat 3.0 software (GraphPad Software Inc., La Jolla,Calif., USA). P values of less than 0.05 are defined as statisticallysignificant.

Example 9 Delivery of Tenofovir to the Brain by Novel Nanovesicles forthe Treatment of Neuro-HIV

In another embodiment, the present disclosure is directed to is todevelopment of a vehicle for the delivery of therapeutic agents into thebrain. In one aspect of this embodiment, the agent is tenofovir. TheExamples below describe the synthesis of a bolaamphiphile with a longhydrophobic domain and a CS head group (GLH-55b), optimization of thetenofovir encapsulation in the vesicles described herein, evaluation ofthe BBB permeability of vesicles that contained GLH-55b in comparison tovesicles that contain GLH-55a, delineation of conditions for detectionof tenofovir in the brain and the concentrations of tenofovir in thebrain following intravenous administration of optimized vesicles withencapsulated tenofovir.

Synthesis of GLH-55b

This compound was prepared as part of an effort to further increase theBBB permeability of the vesicles described herein. It is abolaamphiphile with a CS head group GLH-55b, in which the aliphaticchain has 33 carbon atoms.

Synthesis of the Bolaamphiphile's Skeleton

The synthesis of the asymmetrical skeleton (Scheme 1) of GLH-55b wascarried out through transesterification of methyl vernolate withaliphatic diols using Candida antarctica lipase, immobilized on acrylicresin as the catalyst, to obtain the corresponding mono-hydroxy ester ofvernolic acid. This compound was further reacted with a protecteddicarboxylic acid prepared as described in Scheme 2.

To obtain the monobenzyl ester described in Scheme 2, the dioic acid wasreacted with benzyl alcohol using a ratio of 1:0.6 in toluene underazeotropic conditions, in the presence of catalytic amount of H₂SO₄. Theunreacted dicarboxylic acid was precipitated from the reaction mixtureat room temperature and the product was recrystallized from hexane.

The monobenzyl ester was reacted with thionyl chloride to form thereactive acyl chloride that was subsequently reacted with themonohydroxy vernolate ester in presence of an excess of triethylamine.To avoid epoxide decomposition, the reaction was carried out indichloromethane at −10° C. The crude product was separated by flashchromatography to give the pure hydrophobic skeleton. ESI-MS Calculatedfor C₄₇H₇₈O₇:754.57. Found: 755.2 [M+H⁺], 777.2 [M+Na⁺], 793.1 [M+K⁺].

Attachment of the Head Groups

The two different head groups—CS and ACh—were attached to the longhydrophobic skeleton (about 32-33 atoms) that was obtained as describedin Scheme 1. The attachment of these head groups to the bolaamphiphile'sskeleton is described in Scheme 3

ring. Opening the epoxy ring gave the chloroacetyl group at one of thecarbons of the original epoxy ring and an adjacent hydroxyl group at thesecond carbon of the epoxide. The reaction was performed with a slightexcess of chloroacetic acid, at 100° C. for about 12 h. The solvent wasremoved under reduced pressure and the residue washed with water toremove the excess of the chloroacetic acid. The crude product waspurified by flash chromatography with a mixture of hexane and ethylacetate as the eluent.

ESI-MS: Calculated for C₄₉H₈₁ClO₉: 848.56. Found: 871.3 [M+Na⁺], 887.1[M+K⁺], 731.0 [M+H⁺-H₂O].

The next step was the removal of the benzyl group by hydrogenation in aParr Shaker type hydrogenator. The deprotection of the long chain ester,which was obtained in the previous steps, was performed with hydrogen inthe presence of Pd/C in ethyl acetate as the solvent under pressure of40 psi at room temperature for 2 h. In the FT-IR spectrum it waspossible to detect disappearance of the absorption band characteristicof the aromatic ring at 699 cm⁻¹ and the appearance of the absorptionband at 1706 cm⁻¹, which is characteristic of the carboxylic acid.

The long chain ester, with carboxylic acid at one end was transformedinto an activated acid ester by reacting it with N-hydroxysuccinimide.The new ester has now a good leaving group that can react with amines toform amides. The formation of the long chain activatedN-hydroxysuccinimide ester was performed in the presence ofdicyclohexylcarbodiimide (DCC) at room temperature. The separation ofthe product from the reaction mixture was difficult and therefore asecond column separation was included. After purification on the secondcolumn, HPLC showed that the product is essentially 100% pure. ESI-MSCalculated for C₄₆H₈₀ClNO₁₁:880.6. Found: 880.6 [M+Na⁺], 896.5 [M+K⁺].

The next stage was the attachment of the ACh head group. Theintermediate obtained in the last stage having the chloro acetate groupat one end and the N-hydroxysuccinimide ester at the other end wasreacted with an excess of N,N-diethylaminoethylacetate (that served asthe solvent also) at 40-45° C. under nitrogen for about 7 h. The excessof N,N-diethylaminoethylacetate was removed by repeated additions ofdichloromethane. Argentometric titration of the chloride ion of thequaternary compound gave Cl⁻ 3.14% when the theoretical value was 3.59%,that means that according to Cl⁻ the purity was 87.6%.

The last stage in the synthesis of GLH-55b is the attachment of the lowmolecular weight chitosan (LMWCS) to the bolaamphiphilic skeleton thatnow contains ACh head group on one side of the aliphatic chain and anacylating group (N-hydroxysuccinimide ester) at the other end. Thisconjugation was performed by adding a solution of the intermediatecompound described above in DMSO to a solution of the LMWCS andtriethylamine in DMSO. The molar ratio of the LMWCS to the intermediatecompound with the activated ester was 10:1. The reaction mixture wasstirred for about 70 h at room temperature. The triethylamine wasremoved under reduced pressure and the solution was lyophilized to givea yellow powder.

FT-IR of the product showed that in addition to the absorption peakscharacteristic of the LMWCS, new absorption peaks characteristic ofester, amide and acetate groups at 1739, 1563 and 1241 cm⁻¹ wereobserved. The purity of the bolaamphiphilic product was 75% asdetermined by argentometric titration of the chloride ion. Additionalpurification was necessary to get a purer product.

Optimization of Tenofovir Encapsulation

Methods were established to determine quantitatively the amount of theencapsulated tenofovir based on a UV absorption and determined thepercent of tenofovir encapsulation by various vesicle formulations (datanot shown). Under the conditions that yielded maximum amounts oftenofovir encapsulation, about 18-20% encapsulation was observed. Oneobjective of this work was to achieve the maximum amount of tenofovirencapsulated per vesicle along with a minimum loss of thenon-encapsulated drug. Accordingly, several parameters were examined,such as varying the ratio among the vesicle components, changing theratio of tenofovir to bolaamphiphiles, varying the pH of the hydrationsolution and also adding to the formulation additional additives, in theattempt to increase the encapsulation capacity of the vesicles.

In particular, in order to maximize tenofovir encapsulation thefollowing parameters were studied: (1) tenofovir to GLH ratio; (2)adjusting the concentrations of cholestyrl hemisuccinate (CHEM) andcholesterol (CHOL), components that are used in the vesicle formulation,(3) examining several buffers and pHs in the encapsulation process, (4)time of sonication, and (5) tenofovir concentration, and (6) addition ofstearyl amine to the vesicle formulation to enhance encapsulation bycomplexation with tenofovir.

The results of the Optimization of tenofovir encapsulation, arepresented below in Table 2:

TABLE 2 The effect of the concentration of bolaamphiphiles and tenofoviras well as of the buffer and pH on % tenofovir encapsulation in vesiclesBuffer GLH 19/20 % tenofovir encapsulation per tenofovir and pH of(2:1)* concentration in hydration solution, mg/ml solution mg/ml 1 mg/ml2 mg/ml 5 mg/ml 7.5 mg/ml TRIS pH 8 5 4 TRIS pH 8 10 11 9 6 TRIS pH 8 1524.5 ± 2.1   21 ± 2.1 9 ± 1.4 TRIS pH 8 20 31.4 ± 4.5 22.5 ± 3.5 9.3HEPES pH 10 17.4 **16.5 7.8 3.8 7.5 HEPES pH 15 38.6 18.4, 7.5 **22.5HEPES pH 15 31.2 8 *the standard amount of cholesterol/cholesterylhemisuccinate is 1.6 mg/2.4 mg per 10 mg of the GLH; and **Value for aformulation with ½ the standard amount of cholesterol and cholesterylhemisuccinate.

Note that The numbers in the last 4 columns of Table 2 represent percenttenofovir encapsulation±SEM;

As can be seen from Table 2, a formulation that contained 15 mgbolaamphiphiles and 1 mg tenofovir in HEPES buffer pH 7.5 gave thehighest percent encapsulation (38.6%). The data of Table 2 indicatedthat in order to get maximum amount of encapsulated tenofovir perbolaamphiphiles, it could be advantageous to use more tenofovir perbolaamphiphile. Maximum encapsulation was obtained at a ratio ofencapsulated tenofovir to bolaamphiphile of 0.039 (Table 3, below). Thisfurther suggested that, in order to inject 10 mg/kg of tenofovir itwould be necessary to co-inject 256 mg/kg of GLH. In order to avoid thisrelatively high level of bolaamphiphile, conditions were sought thatwould allow the ratio of encapsulated tenofovir per mg ofbolaamphiphiles to exceed 0.039 (the ratio that had given the maximumencapsulation, as noted above). The values in Table 3, below werecalculated from the values presented in Table 2, above

TABLE 3 Ratio of encapsulated tenofovir to bolaamphiphiles mg tenofovirencapsulated/mg bola Buffer GLH 19/20 per Tenofovir concentration in theand pH of (2:1)*, hydration solution, mg/ml. solution mg/ml 1 mg/ml 2mg/ml 5 mg/ml TRIS pH 8 5 0.008 TRIS pH 8 10 0.011 0.018 0.03 TRIS pH 815 0.016 0.03 0.03 TRIS pH 8 20 0.02 0.022 0.023 HEPES pH 7.5 10 0.0260.024 **0.033 HEPES pH 7.5 15 0.026 **0.033 0.039 HEPES pH 8 15 0.021*The standard amount of cholesterol/cholesteryl hemisuccinate is 1.6mg/2.4 mg per 10 mg of the GLH. **Value for a formulation with ½ thestandard amount of cholesterol and cholesteryl hemisuccinate.The numbers in the 3 last columns of Table 3 represent ratios of mgtenofovir encapsulated mg bolaamphiphile.

In an attempt to increase tenofovir encapsulation, stearyl amine wasadded to determine if that would increase tenofovir encapsulation. Thedata of FIG. 6, depicts the effect of stearyl amine (SA) on tenofovirencapsulation. The vesicles were prepared from formulations with andwithout SA and the non-encapsulated tenofovir (free tenofovir) wasseparated from the encapsulated tenofovir on Sephadex G50 column and theabsorbance at 260 nm of each fraction collected from the column wasread.

As can be seen from FIG. 6, the peak of the encapsulated tenofovirprecedes the peak of the free tenofovir and a progressive reduction inthe non encapsulated tenofovir along with an increase in peak of theencapsulated tenofovir is apparent in vesicles made from formulationsthat contained SA, suggesting SA increase tenofovir encapsulation in thevesicles prepared herein. Notably, the peak of a free tenofovir, withoutvesicles, that was run on the column remains constant.

Since SA increased tenofovir encapsulation, the optimal concentration ofSA that gives maximum tenofovir encapsulation without jeopardizingvesicles stability was examined In these experiments 5 mg/ml tenofovirwas used, and two different concentrations of bolaamphiphiles—10 mg/mland 15 mg/ml GLH19/GLH20 (2:1) and the cholesterol/cholesterylhemisuccinate were in the same concentrations as given in Table 3 above.In addition, Tris buffer to HEPES buffer were compared, with respect totenofovir encapsulation. The results are shown in Table

TABLE 4 Effect of stearyl amine (SA) on tenofovir encapsulation invesicles % Ratio of tenofovir Buffer GLH 19/20 tenofovir encapsulated toand pH of (2:1)*, SA, encapsula- bolaamphiphiles solution mg/ml mg/mltion (mg/mg) TRIS pH 8 10 2.5 12.7 0.064 HEPES pH 7.5 10 2.5 **22 **0.1121.4 0.107 HEPES pH 7.5 15 2.5 18.6 0.08 HEPES pH 7.5 10 1.25 15.9 0.062HEPES pH 7.5 10 3.5 30.9 0.155 HEPES pH 8 15 2.5 23 0.077 *The standardamount of cholesterol/cholesteryl hemisuccinate is 1.6 mg/2.4 mg per 10mg of the GLH. **Value for a formulation with ½ the standard amount ofcholesterol and cholesteryl hemisuccinate used in Table 3.The numbers in the last column of Table represent ratios of mg tenofovirencapsulated/mg bolaamphiphile.

It was found that 2.5 mg/ml stearyl amine effectively increasestenofovir/GLH ratio and the resulting vesicles were stable in atransparent clear solutions, not precipitating with time. The resultsshown in Table 4 show a significant improvement in the mg of tenofovirencapsulated per mg of GLH bola. Thus, in comparison to the resultsshown in Table 3, the ratio of the encapsulated tenofovir to thebolaamphiphiles has been increased from 0.039 to 0.11 and even to 0.155.Formulations that provided the higher ratio of 0.155 provided turbidvesicle solutions in which the vesicle precipitated with time. Inaddition the results of Table 4 show that HEPES buffer gives betterresults in comparison with the Tris buffer. The data of Table 4,suggest, e.g., the ustility of a formulation constituted 10 mg/mlbolaamphiphiles with 2.5 mg/ml SA and HEPES buffer.

Vesicles were prepared from the optimal formulation shown in Table 4with the addition of the GLH-55b, for use in the PK studies. Addition ofthe CS-bolaamphiphile, GLH-55b, did not affect tenofovir encapsulation,nor their size and their zeta potential of those vesicle (Table 5).

TABLE 5 Size and surface charge of vesicles prepared from the indicatedformulations Vesicle formulations contained 10 mg/ml GLH19 + 20(2:1) andstandard concentrations of cholesterol and cholesteryl Radius in nm Zetahemisuccinate determined by DLS potential, mV Empty vesicles 57.19 ±1.14 56.9 ± 2.64 Tenofovir-loaded vesicles 58.37 ± 1.01 47.3 ± 1.31 withSA Tenofovir-loaded vesicles 59.17 ± 1.1   50.2 ± 0.834 with SA and CS(GLH-55b)

Encapsulation may be further improved (without having vesicleprecipitation), by reducing vesicle aggregation, e.g., by: (1)increasing the degree of tenofovir complexation with the bolaamphiphilichead groups by lowering further the anionic additives (cholesterylhemisuccinate); (2) additional time of sonication; (3) the introductionof extrusion or freeze thaw cycles after the sonication steps, and,possibly (4) post loading empty vesicles by a pH gradient across thevesicles.

Ex Vivo and In Vivo Characterization

The Effect of CS Surface Groups on BBB Permeability of Vesicles

Comparison of a bolaamphiphile with the CS head group and a longaliphatic chain (GLH-55b) to a bolaamphiphile with the CS head group anda short aliphatic chain (GLH-55a), was canoed out by evaluating theability of vesicles that contain each of these bolaamphiphile totransport carboxyfluorescein (CF) into the brain via the BBB. CF wasencapsulated in vesicles made from formulations that contained onlyGLH-19 and GLH-20 (vesicles wCS), vesicles that contained CS groupsincorporated by CS-vernolate conjugate and vesicles that contain eitherGLH-55a (vesicles CS bola) or GLH-55b (vesicles CS long bola) andinjected them i.v. into the tail vein of mice. Thirty minutes afterward,the animals we sacrificed and the amount of the CF present in the brainwas measured. The results are shown in FIG. 7. As can be seen in thatfigure, the highest amount of CF was accumulated in the brain of micethat received vesicles that contain the bolaamphiphile with CS headgroup and a long aliphatic chain (GLH-55b), suggesting that thesevesicles contained more CS head groups on their surface.

Determination of Tenofovir in the Brain

As demonstrated above, the vesicles described herein are capable oftransporting encapsulated material into the brain. Accordingly, theability of those vesicles to transport encapsulated tenofovir into thebrain and to determine the concentrations of the transported tenofovirin the brain at various times after the administration were examined.For this purpose an analytical method was required that would be capableof detecting tenofovir in brain homogenates at concentrations similar tothose found in the blood after orally treating human subjects with 300mg tenofovir, a dose that was shown to be effective against HIV. Theblood concentration of tenofovir after treating human subjects with 300mg tenofovir is about 500 ng/ml. Consequently, there was a need for amethod for the determination of tenofovir in brain homogenate at therelevant concentrations. Initial studies led to a method based on HPLCcombined with a UV detector sensitive enough to detect tenofovir atconcentrations in the range of 500 ng/ml. Notably, the determination oftenofovir concentration by this method was done with solutions oftenofovir in aqueous media that contained only a buffer. This method hasbeen refined and to detection of tenofovir spiked into brainhomogenates.

Protein precipitation was accomplished by adding trichloroacetic acid(TCA) at a final concentration of 3.75%, providing a clear supernatantby centrifugation. This supernatant was neutralized by 1% sodiumhydroxide solution and the neutralized extract was examined by HPLC witha UV detector, using an isocratic method with 10% aceto-nitrile and 90%buffer of triethyl amine in water adjusted to pH 5.4 with concentratedphosphoric acid. When applied to a solution of tenofovir in bufferwithout the brain extract, the retention time was 17.3 min (FIG. 8 PanelA). However, when applied to a supernatant of the brain homogenate afterremoval of the proteins with TCA, without tenofovir, various peaks wereobserved with different retention times (Rt) between 2.5-23 minutes. Thebroad peak at 22.5 min overlapped the peak of the tenofovir at 17 min(compare FIG. 8 Panel B to FIG. 8 Panel A) and would interfere with thedetermination of tenofovir concentration in the extract. To narrow thepeak of the endogenous compound with the Rt of 22.5 min, a solid phaseextraction (SPE) method was employed, using BOND ELUT-C18 column. Afterthe treatment with SPE some peaks disappeared and the others becamenarrower. Under the isocratic conditions consisting of 0.3% acetonitrileand 0.1% acetic acid in water, the retention time of Tenofovir was about17 minutes and the chromatogram of the supernatant after SPE treatmentshowed no peaks in this part (FIG. 8 Panel C).

Under these conditions, tenofovir was well separated from the endogenouspeaks and it showed an isolated measurable peak, as illustrated in FIG.9).

As demonstrated above, conditions for the determination of tenofovirconcentrations in brain extract had been established. Differentconcentrations of tenofovir were then spiked into a supernatant obtainedafter deproteinization of brain homogenate with TCA and centrifugation.The supernatant with the tenofovir was loaded into the SPE cartridge,which was conditioned by 3 ml methanol and 3 ml of 150 mM ammoniumacetate (pH=5.0). After washing with 900 μl of 100 mM ammonium acetate(pH=7.0), in order to obtain less interference at the retention time ofeach component, the column was eluted by 500 μl of methanol to collectthe tenofovir. The solvent was removed under nitrogen at 60° C. Theextracted sample was dissolved into 120 μl of water and methanolsolution (90:10, v/v) and injected into the HPLC. The data obtained arepresented in FIG. 10.

Calculation of the area under the peak of the tenofovir data of FIG. 10,allowed construction of the calibration curve set forth in FIG. 11,which showed a linear relationship between the tenofovir concentrationsand the area under the peaks for these concentrations.

The calibration curve obtained from the areas under the peaks incomparison to the areas under the peaks for the chromatogram oftenofovir that was spiked into brain extract obtained afterprecipitation of the proteins is shown in FIG. 13.

From these calibration curves it can be seen that lower amounts oftenofovir have been detected when Tenofovir was spiked into brainhomogenate (FIG. 13, curve B) compared to samples in which the tenofovirwas spiked into the supernatant obtained by deproteinization of thebrain homogenate (FIG. 13, curve A). When the percent recovery oftenofovir was calculated for each concentration, it was found that forthe lower tenofovir concentrations (6.25 and 12.5 μg/ml), the recoverywas between 36-38% and for the highest concentration that was tested (25μg/ml), the recovery was 54% (Table 6). These data indicated that acalibration curve will have to be done after knowing the concentrationrange that was found in the brain according to the areas under the peaksthat will be obtained from homogenates of brains taken from animals thatwere injected with tenofovir-loaded vesicles.

TABLE 6 Percent recovery of tenofoivir from brain homogenates Tenofovirconc. Area under the peak (μg/ml) A B Percent recovery 6.25 185 66 35.712.50 376 144 38.3 25.00 651 353 54.2 A = tenofovir concentrations afterspiking the drug into brain extract obtained after proteinprecipitation; B = tenofovir concentrations after spiking the drug intobrain homogenates

The experiments above used higher concentrations of tenofovir than thoseexpected to be found in the brain after delivering the drug encapsulatedin vesicles. This was done in order to provide a clear signal to noiseratio during this, preliminary, developmental work. Lower concentrationsof tenofovir concentrations can be detected using with the HPLC methoddescribed herein or methods employing LC-MS. However, the assaydescribed herein, using higher concentrations of tenofovir, as usefuldetecting tenofovir in the brain after injecting mice i.v. withtenofovir-loaded vesicles.

Determination of Tenofovir Concentrations in the Brain after InjectingAnimals with Tenofovir-Loaded Vesicles.

In view of the conditions for the determination of tenofovir in brainhomogenates established herein, mice were injected with 7.5 mg/kg oftenofovir encapsulated in vesicles prepared as for Table 5 above thatcontained the optimal GLH-55b and SA and brain homogenates were preparedfrom the brains of these mice for the determination of tenofovirconcentrations. The chromatograms of the brain extracts from these miceshowed peaks with retention times that correspond to tenofovir, asdepicted in FIG. 14 C. The peaks in the chromatograms observed (FIG. 14C). and using the lowest point of the calibration curves obtained fortenofovir spiked into brain homogenate (curve B in FIG. 13), it wasestimated that the concentration of tenofovir in the brain of the animalthat received 7.5 mg/kg encapsulated tenofovir to be 22.7 μg/gr brain.This concentration value is significant and should have a therapeuticaffect against HIV.

A subsequent in vivo experiment was carried out as above but withtesting over different periods of time from of 15 min, 30 min, 60 minand 120 min after IV injection the amount of tenofovir was in microgr/grm brain 6.2, 2.2, 1.0 and 0.5 respectively. While the freetenofovir injected had levels of 0 less than 0.2 micrograms/gr brain.Thus indicating the efficacy of the vesicles to delivery tenofovir tothe brain.

As described here a novel formulations of bolavesicles can be producedthrough co-assembly of HIV drugs with bolaamphiphile/lipid unilamellarvesicles. The formulations can be examined for their chemical andbiophysical properties. In one embodiment the vesicles formed from thebolaamphiphiles by aggregation contain additives that help to stabilizethe vesicles, by stabilizing the vesicle's membranes, such as but notlimited to cholesterol derivatives such as cholesteryl hemisuccinate andcholesterol itself and combinations such as cholesteryl hemisuccinateand cholesterol. In still another embodiments the vesicles in additionto these components and the bolamphiphiles have another additives whichdecorates the outer vesicle membranes with groups or pendants thatenhance penetration though biological barriers such as the BBB, orgroups for targeting to specific sites. Further the bolaamphiphile headcan interact with the active agents to be delivered such as tenofovir byionic interactions to enhance the % encapsulation via complexation andwell as passive encapsulation within the vesicles core. Further theformulation may contain other additives such as stearyl amine within theveicles membranes to further enhance the degree of encapsulation ofactive agents like tenofovir. To maximize the ionic interactions betweenthe components to increase the efficiency of encapsulation the pH of theaqueous hydrating solutions can be optimized by well known methods ofthe state of art.

The incorporation of HIV drug within the bolavesicles is shown tosignificantly modulate interactions with membrane bilayers in modelsystems. This observation is important, suggesting that HIV drugsencapsulated in bolavesicles might be excellent candidates for targetingand transport of different molecular cargoes into the brain.

LC-MS Method for the Determination of Tenofovir in the Brain

The chromatographic separation was performed with an HPLC Agilent 1100instrument and Auto-sampler G 1329A ALS 1200 Series with Frizzier G1330BFC/ALS Therm, at a temperature of 4° C., using a reverse phase Kromasile100A C18 250×2.0 (5 μm) column at 30° C. under gradient conditions. Themobile phase consisted of two solvents: (A) acetonitrile and (B) 0.5%formic acid in water and was operated under the following conditions:

TABLE 7 Percent Percent Time (min) Solvent A Solvent B 0 10 90 7 10 9012 100 0 15 100 0 22 10 99 30 10 90

The MS/MS detector was an Ion Trap MS Esquire 3000 Plus (BrukerDaltonics), operating in the ESI positive polarity mode. The internalstandard was 2′-deoxyadenosine 5′-phosphate.

The MS peaks that were obtained from these compounds (tenofovir and2′-deoxyadenosine 5′-phosphate) are shown in FIG. 16.

After establishing conditions for the LC-MS as described above, brainhomogenate and blood samples were prepared and spiked knownconcentrations of tenofovir and a fixed concentration of the internalstandard into the blood samples and the brain homogenates. The specimenswere then processed by adding 180 μL of 10% Tri Chloro Acetic acid (TCA)to 300 μL of the homogenate followed by centrifugation to obtain a clearsolution of blood or brain extracts. The supernatant that was obtainedby the centrifugation was neutralized with 200 μL of 1% NaOH. The MSsignals that were obtained from each sample were plotted as a functionof the spiked tenofovir concentration to obtain calibration curves forthe blood (FIG. 17A) and for the brain (FIG. 17B).

As can be seen from FIG. 17, tenofovir concentrations as low as 50 ngcould have been measured accurately by this method and, when lowerconcentrations in tissue extracts were obtained, the actualconcentration was calculated by an extrapolation, as the calibrationcurve was linear for the relevant concentration range.

Pharmacokinetic Studies

For the PK studies, vesicles optimized as described above were used forthe delivery of tenofovir into the brain. This formulation contains in 1mL:10 mg GLH 19 and GLH 20 in a ratio of 2/1, 1 mg GLH-55b, 2.4 mgcholesteryl hemisuccinate, 1.6 mg cholesterol, 2.5 mg stearyl amine, and5 mg tenofovir. The vesicles were prepared by film hydration followed bysonication using a HEPES hydration buffer. The vesicles size that wasobtained with this optimal formulation was ˜100 nm in diameter, thevesicles were positively charged with a zeta potential of about 30 mVand 20% of the added tenofovir was encapsulated under these conditions.

In the first set of the PK experiments, mice (average weight was 22 gper mouse) were injected with vesicles that contained encapsulatedtenofovir or with empty vesicles followed by free tenofovir (4 mice pergroup). Since during the encapsulation procedure some of the tenofovirremains non-encapsulated (the amount of the non-encapsulated tenofovirdepends on the encapsulation conditions, as has been shown in previousstudies and in this particular case the amount of the encapsulatedtenofovir was 1 mg/mL vesicles) and the vesicles were not purifiedbefore injection (to avoid dilution of the vesicles that occur duringthe purification process), the dose of the tenofovir was adjusted sothat in each case an equal dose of total tenofovir was injected. Thedose of the total tenofovir was 34 mg/kg, out of which 6.8 mg/kg wasencapsulated (only when encapsulated tenofovir was injected). This doseof encapsulated tenofovir is equivalent to about 475 mg per humansubject of an average weight of 70 Kg, which is within the dose rangegiven to AIDS patients. Before the injection of the test material, micewere pretreated with 0.5 mg/kg pyridostigmine to inhibit cholineesterases in the blood (unless otherwise stated), since vesicles thatcontain GLH-20 release their content in presence of choline esterases.At various times after the injection (15, 30, 75, 120 and 240 min),blood samples were withdrawn and the mice were perfused with 10 mL PBSand sacrificed. Brains were immediately removed after sacrificing themice and brain homogenates were prepared by homogenizing the brains inPBS (4 mL PBS per gram brain). Extracts of the homogenates were thenprepared by adding 180 μL of 10% TCA to 300 μL of the homogenatefollowed by centrifugation to obtain a clear solution of the brainextract. The supernatant that was obtained by the centrifugation wasneutralized with 200 μL of 1% NaOH. Tenofovir concentration in the clearbrain extract was determined by LC-MS, using the calibration curve whichis described above in FIG. 17. The blood samples were centrifuged toseparate the serum from the blood cells and processed in the same way asthe brain homogenates. Tenofovir concentration was determined by LC-MS.

The results of the tenofovir concentrations in the blood at varioustimes after the injection of the test material are shown in FIG. 18.

As can be seen from FIG. 18, the concentration of the blood tenofovirdropped quickly after the injection of the test material. The half-lifeof tenofovir in the blood was calculated to be about 20 min. Theconcentrations of tenofovir in the brain was determined in brainextracts prepared from the same animals, which were used to obtain thedata for FIG. 18 and the results are shown in FIG. 19.

From the results in FIG. 19, it can be seen that, when the mice werepretreated with pyrido, the half-life of tenofovir in the brain waslonger than the half-life of tenofovir in the blood (compare data fromFIG. 18 with data that were obtained with the same animals and that arepresented in FIG. 19). Thus, the apparent half-life of tenofovir in thebrain was about 40 min as compared to 20 minutes half-life of tenofovirin the blood of the same animals. The apparent half-life of tenofovir inthe blood reflects both free tenofovir and encapsulated tenofovir, asthe non-encapsulated tenofovir was not removed from the vesicles beforethe injection. Since the encapsulated tenofovir constituted only 20% ofthe injected tenofovir, the data indicate that the actual half-life ofthe encapsulated tenofovir in the blood is longer than 20 min. Bycomparison, in the brain, only encapsulated tenofovir enters into thisorgan, and the apparent half-life in the brain is likely identical tothe actual half-life and it may be a reflection of the actual half-lifeof encapsulated tenofovir in the blood. When the animals were notpretreated with pyrido, the half-life in the brain was similar to thehalf-life of tenofovir in the blood, suggesting that most of thevesicles were opened in the blood by the blood choline esterases thatwere not inhibited by pyrido. Tenofovir encapsulated in GLH-19 vesiclesdid not persist long in the brain, in part because tenofovir was notreleased efficiently in the brain from vesicles that are not opened byAChE. The initial high concentration of tenofovir in the brain inanimals that were injected with tenofovir encapsulated in GLH-19vesicles, which are not opened by choline esterases, is probably due tothe presence of encapsulated tenofovir in the brain a short time afterthe injection. Since tenofovir was not released in the brain from GLH-19vesicles, it did not accumulate in the brain as in the case whentenofovir was encapsulated in vesicles made of a mixture of GLH-19 andGLH-20, which are opened and release their content in presence of AChE.The GLH-19 vesicles apparently were circulated back out of the brainwithout releasing their content in the brain.

In the second set of the PK studies, all the mice were pretreated withpyrido 15 min prior to the injection of 7.5 mg/kg tenofovir encapsulatedin optimal vesicles or with 75 mg/kg free tenofovir with no vesicles.The results that show the concentrations of tenofovir in the brain atvarious times after the injection are shown in FIG. 20.

As can be seen from FIG. 20, the concentration of tenofovir in the brainafter delivering the drug encapsulated in the optimized V-Smart™vesicles was above the therapeutic threshold (which is estimated to be100 ng/mL) at all the time points (up to 2 hours). After 15 min, theconcentration of the tenofovir in the brain was higher than 6 microgramsper gram brain and, even at 60 min after the injection, the encapsulatedtenofovir's concentration in the brain was about 1 microgram per mL,still one order of magnitude higher than the therapeutic levels andcomparable to the blood level at this time point. The relatively rapiddrop of tenofovir concentration in the brain may be explained byintracellular metabolism into the active metabolites of the drug, whichare mono or dephosphorylated derivatives of tenofovir. An alternativepossibility is that the brain pumps out tenofovir that was released inthe brain from the vesicles, most probably by the PGp pump, which is afunctional component of the BBB. Since the vesicles disclosed above havegood cellular uptake, it appears that the rapid reduction in tenofovirconcentrations in the brain is mostly due to intracellular metabolisminto the active metabolites and only a small amount of tenofovir isremoved from the brain by the Pgp pump. By comparison to encapsulatedtenofovir, when the free drug was injected at a dose twice of theencapsulated dose, the concentrations of tenofovir in the brain werevery low, below the measurable level and on the border of the detectionlimit, which is 40 ng/ml (FIG. 20). These results clearly show that ourV-Smart™ vesicles are capable of delivering into the brain significantamounts of tenofovir, a drug that in its free form does not penetrateinto the brain almost at all. These results indicate that the V-Smart™vesicles described above, which were optimized for the delivery oftenofovir into the brain, may be beneficial in the treatment ofneuro-HIV.

From the foregoing description, various modifications and changes in thecompositions and methods provided herein will occur to those skilled inthe art. All such modifications coming within the scope of the appendedclaims are intended to be included therein.

All publications, including but not limited to patents and patentapplications, cited in this specification are herein incorporated byreference as if each individual publication were specifically andindividually indicated to be incorporated by reference herein as thoughfully set forth.

At least some of the chemical names of compounds of the invention asgiven and set forth in this application, may have been generated on anautomated basis by use of a commercially available chemical namingsoftware program, and have not been independently verified.Representative programs performing this function include the Lexichemnaming tool sold by Open Eye Software, Inc. and the Autonom Softwaretool sold by MDL, Inc. In the instance where the indicated chemical nameand the depicted structure differ, the depicted structure will control.

Chemical structures shown herein were prepared using ISIS®/DRAW. Anyopen valency appearing on a carbon, oxygen or nitrogen atom in thestructures herein indicates the presence of a hydrogen atom. Where achiral center exists in a structure but no specific stereochemistry isshown for the chiral center, both enantiomers associated with the chiralstructure are encompassed by the structure.

REFERENCES

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Expert Rev Neurother. 6(10):1495-509.-   *Popov M., Linder C., Deckelbaum R. J., Grinberg S., Hansen I. H.,    Shaubi E., Waner T., Heldman E. (2009) Cationic vesicles from novel    bolaamphiphilic compounds. J Liposome Res. 20(2):147-159.-   *Popov M, Grinberg S, Linder C, Bachar Z, Waner T, Deckelbaum R,    Heldman E. (2011) Site-directed decapsulation of bolaamphiphilic    vesicles with enzymatic cleavable surface groups submitted to the    Journal of Controlled Release.-   *Puri, A., Loomis, K., Smith, B., Lee, J., Yavlovich, A.,    Heldman, E. and Blumenthal, R. (2009) Lipid-Based Nanoparticles as    Pharmaceutical Drug Carriers: From Concepts to Clinic. Crit Rev Ther    Drug Carrier Syst, 26(6): 523-580.-   Saiyed Z, Gandhi N, and Nairi M (2010)Magnetic Nanoformulation of    Azidothymidine 5′-triphosphate for Targeted Delivery across the    Blood-Brain Barrier. International Journal of Nanomedicine 5:157-166-   Songjiang Z and Lixiang W. (2009) Amyloid-Beta Associated with    Chitosan Nano-Carrier has Favorable Immunogenicity and Permeates the    BBB. AAPS Pharm Sci Tech, 10(3):900-905.-   Spudich S and Antses B (2011) Central Nervous System Complications    of HIV Infection. Top. Antiviral Med 19(2), 48-57.-   Stern J, Freisleben H J, Janku S, Ring K. (1992) Black lipid    membranes of tetraether lipids from Thermoplasma acidophilum,    Biochim Biophys Acta 1128:227-236.-   Varatharajan L and Thomas S. (2009) The transport of anti-HIV drugs    across blood-CNS interfaces: Summary of current knowledge and    recommendations for further Research Antiviral Res. 2009 May; 82(2):    A99-A109.-   *Wiesman Z., Dom N. B., Sharvit E., Grinberg S., Linder C., Heldman    E., Zaccai M. (2007) Novel cationic vesicle platform derived from    vernonia oil for efficient delivery of DNA through plant cuticle    membranes. J. 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1. A pharmaceutical composition or a formulation comprising abolaamphiphile complex or nano-sized vesicles; wherein thebolaamphiphile complex or nano-sized vesicles comprises one or morebolaamphiphilic compounds and a compound active against HIV, wherein thebolaamphiphilic compound is a compound according to formula 1:HG²-L¹-HG¹  I or a pharmaceutically acceptable salt, solvate, hydrate,prodrug, stereoisomer, tautomer, isotopic variant, or N-oxide thereof,or a combination thereof: wherein: each HG¹ and HG² is independently ahydrophilic head group; and L¹ is alkylene, alkenyl, heteroalkylene, orheteroalkenyl linker; unsubstituted or substituted with C₁-C₂₀ alkyl,hydroxyl, or oxo; and a pharmaceutically-acceptable carrier.
 2. A methodof delivering HIV active drugs into animal or human brain comprising thestep of administering to the animal or human a pharmaceuticalcomposition or a formulation comprising a bolaamphiphile complexaccording to claim
 1. 3. (canceled)
 4. (canceled)
 5. The pharmaceuticalcomposition according to claim 1, wherein L¹ is heteroalkylene, orheteroalkenyl linker comprising C, N, and O atoms; unsubstituted orsubstituted with C₁-C₂₀ alkyl, hydroxyl, or oxo.
 6. The pharmaceuticalcomposition according to claim 1, wherein L¹ is—O—(CH₂)_(n1)—O—C(O)—(CH₂)_(n2)—C(O)—O—(CH₂)_(n3)—O—.
 7. Thepharmaceutical composition according to claim 1, wherein L is—O-L²-C(O)—O—(CH₂)_(n4)—O—C(O)-L³-O—, or—O-L²-C(O)—O—(CH₂)_(n5)—O—C(O)—(CH₂)_(n6)—, and wherein each L² and L³is C₄-C₂₀ alkylene or alkenyl linker; unsubstituted or substituted withC₁-C₈ alkyl or hydroxy; and n4, n5, and n6 is independently an integerfrom 4-20.
 8. The pharmaceutical composition according to claim 7,wherein each L² and L³ is independently—C(R¹)—C(OH)—CH₂—(CH═CH)—(CH₂)_(n7)—; R¹ is C₁-C₈ alkyl, and n7 isindependently an integer from 4-20.
 9. The pharmaceutical compositionaccording to claim 1, wherein L¹ is

wherein: each Z¹ and Z² is independently —C(R³)₂—, —N(R³)— or —O—; eachR^(1a), R^(1b), R³, and R⁴ is independently H or C₁-C₈ alkyl; eachR^(2a) and R^(2b) is independently H, C₁-C₈ alkyl, OH, or alkoxy; eachn8, n9, n11, and n12 is independently an integer from 1-20; n10 is aninteger from 2-20; and each dotted bond is independently a single or adouble bond. and wherein each methylene carbon is unsubstituted orsubstituted with C₁-C₄ alkyl; and each n1, n2, and n3 is independentlyan integer from 4-20.
 10. The pharmaceutical composition according toclaim 1, wherein the bolaamphiphilic compound is a compound according toformula II, III, IV, V, or VI:

or a pharmaceutically acceptable salt, solvate, hydrate, prodrug,stereoisomer, tautomer, isotopic variant, or N-oxide thereof, or acombination thereof; wherein: each HG¹ and HG² is independently ahydrophilic head group; each Z¹ and Z² is independently —C(R³)₂—,—N(R³)— or —O—; each R^(1a), R^(1b), R³, and R⁴ is independently H orC₁-C₈ alkyl; each R^(2a) and R^(2b) is independently H, C₁-C₈ alkyl, OH,alkoxy, or O-HG¹ or O-HG²; each n8, n9, n11, and n12 is independently aninteger from 1-20; n10 is an integer from 2-20; and each dotted bond isindependently a single or a double bond.
 11. (canceled)
 12. (canceled)13. (canceled)
 14. The pharmaceutical composition according to claim 10,wherein the bolaamphiphilic compound is a compound according to formulaII, III, IV, V, or VI; and each n8 and n12 is independently 1, 2, 3, or4.
 15. (canceled)
 16. The pharmaceutical composition according to claim10, wherein the bolaamphiphilic compound is a compound according toformula II, III, IV, V, or VI; and each R^(2a) and R^(2b) isindependently H, OH, alkoxy, or O-HG¹ or O-HG².
 17. (canceled) 18.(canceled)
 19. The pharmaceutical composition according to claim 10,wherein the bolaamphiphilic compound is a compound according to formulaII, III, IV, V, or VI; and each R^(1a) and R^(1b) is independently H,Me, Et, n-Pr, i-Pr, n-Bu, i-Bu, sec-Bu, n-pentyl, isopentyl, n-hexyl,n-heptyl, or n-octyl.
 20. (canceled)
 21. (canceled)
 22. (canceled) 23.The pharmaceutical composition according to claim 10, wherein thebolaamphiphilic compound is a compound according to formula II, III, IV,or V; n10 is an integer from 2-16.
 24. (canceled)
 25. (canceled)
 26. Thepharmaceutical composition according to claim 10, wherein thebolaamphiphilic compound is a compound according to formula VI; and R⁴is H, Me, Et, n-Pr, i-Pr, n-Bu, i-Bu, sec-Bu, n-pentyl, or isopentyl.27. (canceled)
 28. The pharmaceutical composition according to claim 10,wherein the bolaamphiphilic compound is a compound according to formulaII, III, IV, V, or VI; and each Z¹ and Z² is independently C(R³)₂— or—N(R³)—.
 29. The pharmaceutical composition according to claim 10,wherein the bolaamphiphilic compound is a compound according to formulaII, III, IV, V, or VI; and each Z¹ and Z² is independently C(R³)₂—, or—N(R³)—; and each is independently H, Me, Et, n-Pr, i-Pr, n-Bu, i-Bu,sec-Bu, n-pentyl, or isopentyl.
 30. (canceled)
 31. The pharmaceuticalcomposition according to claim 10, wherein the bolaamphiphilic compoundis a compound according to formula II, III, IV, V, or VI; and each Z¹and Z² is —O—.
 32. The pharmaceutical composition according to claim 10,wherein the bolaamphiphilic compound is a compound according to formulaII, III, IV, V, or VI; and each HG¹ and HG² is independently selectedfrom:

wherein: X is —NR^(5a)R^(5b), or —N⁺R^(5a)R^(5b)R^(5c); each R^(5a), andR^(5b) is independently H or substituted or unsubstituted C₁-C₂₀ alkylor R^(5a) and R^(5b) may join together to form an N containingsubstituted or unsubstituted heteroaryl, or substituted or unsubstitutedheterocycle; each R^(5c) is independently substituted or unsubstitutedC₁-C₂₀ alkyl; each R⁸ is independently H, substituted or unsubstitutedC₁-C₂₀ alkyl, alkoxy, or carboxy; m1 is 0 or 1; and each n13, n14, andn15 is independently an integer from 1-20.
 33. (canceled)
 34. (canceled)35. (canceled)
 36. (canceled)
 37. (canceled)
 38. The pharmaceuticalcomposition according to claim 1, wherein the bolaamphiphilic compoundis a compound according to formula VIIa, VIIb, VIIc, or VIId:

or a pharmaceutically acceptable salt, solvate, hydrate, prodrug,stereoisomer, tautomer, isotopic variant, or N-oxide thereof, or acombination thereof; wherein: each X is —NR^(5a)R^(5b), or—N⁺R^(5a)R^(5b)R^(5c); each R^(5a), and R^(5b) is independently H orsubstituted or unsubstituted C₁-C₁₀ alkyl or R^(5a) and R^(5b) may jointogether to form an N containing substituted or unsubstitutedheteroaryl, or substituted or unsubstituted heterocycle; each R^(5c) isindependently substituted or unsubstituted C₁-C₂₀ yl; n10 is an integerfrom 2-20; and each dotted bond is independently a single or a doublebond.
 39. The pharmaceutical composition according to claim 1, whereinthe bolaamphiphilic compound is a compound according to formula VIIIa,VIIIb, VIIIc, or VIIId:

or a pharmaceutically acceptable salt, solvate, hydrate, prodrug,stereoisomer, tautomer, isotopic variant, or N-oxide thereof, or acombination thereof; wherein: each X is —NR^(5a)R^(5b), or —N^(+R)^(5a)R^(5b)R^(5c); each R^(5a), and R^(5b) is independently H orsubstituted or unsubstituted C₁-C₂₀ alkyl or R^(5a) and R^(5b) may jointogether to form an N containing substituted or unsubstitutedheteroaryl, or substituted or unsubstituted heterocycle; each R^(5c) isindependently substituted or unsubstituted C₁-C₂₀ alkyl; n10 is aninteger from 2-20; and each dotted bond is independently a single or adouble bond.
 40. The the pharmaceutical composition according to claim1, wherein the bolaamphiphilic compound is a compound. according toformula IXa, IXb, or IXc:

or a pharmaceutically acceptable salt, solvate, hydrate, prodrug,stereoisomer, tautomer, isotopic variant, or N-oxide thereof, or acombination thereof; wherein: each X is —NR^(5a)R^(5b), or—N⁺R^(5a)R^(5b)R^(5c); each R^(5a) and R^(5b) is independently H orsubstituted or unsubstituted C₁-C₂₀ alkyl or R^(5a) and R^(5b) may jointogether to form an N containing substituted or unsubstitutedheteroaryl, or substituted or unsubstituted heterocycle; each R^(5c) isindependently substituted or unsubstituted C₁-C₂₀ alkyl; n10 is aninteger from 2-20; and each dotted bond is independently a single or adouble bond.
 41. The pharmaceutical composition according to claim 1,wherein the bolaamphiphilic compound is a compound according to formulaXa, Xb, or Xc:

or a pharmaceutically acceptable salt, solvate, hydrate, prodrug,stereoisomer, tautomer, isotopic variant, or N-oxide thereof, or acombination thereof; wherein: each X is —NR^(5a)R^(5b), or—N⁺R^(5a)R^(5b)R^(5c); each R^(5a) and R^(5b) is independently H orsubstituted or unsubstituted alkyl or R^(5a) and R^(5b) may jointogether to form an N containing substituted or unsubstitutedheteroaryl, or substituted or unsubstituted heterocycle; each R^(5c) isindependently substituted or unsubstituted C₁-C₂₀ alkyl; n10 is aninteger from 2-20; and each dotted bond is independently a single or adouble bond.
 42. (canceled)
 43. (canceled)
 44. The pharmaceuticalcomposition according to claim 38, wherein the bolaamphiphilic compoundis a compound according to formula VIIa-VIId, VIIIa-VIIId, IXa-IXc, orXa-Xc; n10 is an integer from 2-16.
 45. (canceled)
 46. (canceled) 47.The pharmaceutical composition according to claim 32, wherein eachR^(5a), R^(5b), and R^(5c) is independently substituted or unsubstitutedC₁-C₂₀ alkyl.
 48. (canceled)
 49. (canceled)
 50. The pharmaceuticalcomposition according to claim 32, wherein two of R^(5a), R^(5b) andR^(5C) are independently C₁-C₂₀ alkyl substituted with —OC(O)R⁶; and R⁶is C₁-C₂₀ alkyl.
 51. The pharmaceutical composition according to claim32, wherein one of R^(5a), R^(5b), and R^(5c) is C₁-C₂₀ alkylsubstituted with —OC(O)R⁶; and R⁶ is Me, Et, n-Pr, i-Pr, n-Bu, i-Bu,sec-Bu, n-pentyl, isopentyl, n-hexyl, n-heptyl, or n-octyl.
 52. Thepharmaceutical composition according to claim 32, wherein one of R^(5a),R^(5b) and R^(5c) is C₁-C₁₀ alkyl substituted with amino, alkylamino ordialkylamino.
 53. (canceled)
 54. The pharmaceutical compositionaccording to claim 32, wherein R^(5a), and R^(5b) together with the Nthey are attached to form substituted or unsubstituted heteroaryl. 55.(canceled)
 56. The pharmaceutical composition according to claim 32,wherein R^(5a), and R^(5b) together with the N they are attached to formsubstituted or unsubstituted monocyclic or bicyclic heterocycle. 57.(canceled)
 58. (canceled)
 59. (canceled)
 60. (canceled)
 61. (canceled)62. The pharmaceutical composition according to claim 32, wherein X is achitosanyl group.
 63. The pharmaceutical composition according to claim1, wherein the bolaamphiphilic compound is a pharmaceutically acceptablesalt.
 64. (canceled)
 65. (canceled)
 66. The pharmaceutical compositionaccording to claim 1, wherein the bolaamphiphilic compound is any one ofthe bolaamphilic compounds listed in Table
 1. 67. (canceled)
 68. Thepharmaceutical composition of claim 1 wherein the carrier is aparenteral carrier.
 69. The pharmaceutical formulation of claim 1comprising a bolaamphilic compound according to formula I-Xc. 70.(canceled)
 71. The pharmaceutical formulation of claim 1 comprisingbolaamphilic vesicles comprising one or more bolaamphilic compoundsaccording to formula I-Xc and a compound active against HIV. 72.(canceled)
 73. The pharmaceutical formulation of claim 1 comprising anano-particle comprising one or more bolaamphiphilic compounds and acompound active against HIV.
 74. The nano-particle according to claim73, wherein the bolaamphiphilic compounds and a compound active againstHIV are encapsulated within the nano-particle.
 75. (canceled)
 76. Thepharmaceutical composition of claim 1 wherein the HIV active drug isTenofovir or({[(2R)-1-(6-amino-9H-purin-9-yl)propan-2-yl]oxy}methyl)phosphonic acid.77. The pharmaceutical composition claim 1, wherein the HIV active drugis fosamprenavir, enfuvirtide, saquinavir, lamivudine or stavudine. 78.A method for treatment or diagnosis of diseases or disorders selectedfrom HIV and related diseases comprising administering to a patient inneed thereof an effective amount of a pharmaceutical compositionaccording to claim
 1. 79. The pharmaceutical composition according toclaim 1, wherein the vesicles comprise an additive that increasesencapsulation of a therapeutic agent.
 80. (canceled)
 81. (canceled) 82.(canceled)
 83. The pharmaceutical composition of claim 1 wherein thevesicles are formed from the bolaamphiphiles by aggregation contain alsoadditives that help to stabilize the vesicles, by stabilizing thevesicle's membranes, the additive being a cholesterol derivative,cholesteryl hemisuccinate, cholesterol and combinations thereof.
 84. Thepharmaceutical composition of claim 83 wherein the vesicles furthercomprise additives that decorate the outer vesicle membranes with groupsor pendants that enhance penetration though biological barriers such asthe BBB, or groups for targeting to specific sites.
 85. Thepharmaceutical composition of claim 83 wherein the bolaamphiphile headcan interact with the active agents to be delivered such as tenofovir byionic interactions to enhance the % encapsulation via complexation andwell as passive encapsulation within the vesicles core.
 86. Thepharmaceutical composition of claim 85 wherein the vesicle furthercontains additives within the vesicles' membranes that further enhancethe degree of encapsulation of active agents.
 87. The method of claim 2wherein the HIV active drug is Tenofovir or({[2R)-1-(6-amino-9H-purin-9-yl)propan-2-yl]oxy}methyl)phosphonic acid.88. The method of claim 2 wherein the HIV active drug is fosamprenavir,enfuvirtide, saquinavir, lamivudine or stavudine.
 89. The method ofclaim 78 wherein the HIV active drug is Tenofovir or({[2R)-1-(6-amino-9H-purin-9-yl)propan-2-yl]oxy}methyl)phosphonic acid.90. The method of claim 78 wherein the HIV active drug is fosamprenavir,enfuvirtide, saquinavir, lamivudine or stavudine.
 91. The pharmaceuticalcomposition of claim 1, wherein the bolaamphiphilic compound is acompound according to any of formula VIIa, VIIb, VIIc, VIId, VIIIa,VIIIb, VIIIc, VIIId, IXa, IXb, IXc, Xa, Xb, and Xc, wherein the —OHgroup adjacent to the head group, is absent.